The human papillomavirus (HPV) minor capsid protein L2 is a promising candidate for a broadly protective HPV vaccine yet the titers obtained in most experimental systems are rather low. Here we examine the potential of empty AAV2 particles (AAVLPs), assembled from VP3 alone, for display of L2 epitopes to enhance their immunogenicity. Insertion of a neutralizing epitope (amino acids 17–36) from L2 of HPV16 and HPV31 into VP3 at positions 587 and 453, respectively, permitted assembly into empty AAV particles (AAVLP(HPV16/31L2)). Intramuscularly vaccination of mice and rabbits with AAVLP(HPV16/31L2)s in montanide adjuvant, induced high titers of HPV16 L2 antibodies as measured by ELISA. Sera obtained from animals vaccinated with the AAVLP(HPV16/31L2)s neutralized infections with several HPV types in a pseudovirion infection assay. Lyophilized AAVLP(HPV16/31L2) particles retained their immunogenicity upon reconstitution. Interestingly, vaccination of animals that were pre-immunized with AAV2 - simulating the high prevalence of AAV2 antibodies in the population - even increased cross neutralization against HPV31, 45 and 58 types. Finally, passive transfer of rabbit antisera directed against AAVLP(HPV16/31L2)s protected naïve mice from vaginal challenge with HPV16 pseudovirions. In conclusion, AAVLP(HPV16/31L2) particles have the potential as a broadly protective vaccine candidate regardless of prior exposure to AAV.
Persistent high-risk human papillomavirus (HPV) infection has been linked to the development of cervical cancer [1] . Attempts to prevent infection with the most prevalent high risk HPV types – HPV16 and HPV18 – led to the development of two highly efficacious HPV vaccines that comprise virus-like particles (VLPs) assembled from the major capsid protein L1. However these vaccines target only two of the 15 high-risk HPV types, responsible for 70–80% of cervical cancer cases. The prevention of 96% of cervical cancer would require the immunity against at least 7 high-risk HPV types (HPV16, 18, 31, 33, 45, 52 and 58), but this increases the cost and complexity of manufacture [2] . Future vaccines should therefore attempt to extend immunization against other high-risk HPV types. For broad application also in developing countries they should be provided inexpensively in a temperature stable formulation.
The minor capsid protein L2 harbors several regions that can be targeted by neutralizing antibodies [3] – [5] . One of which, located at amino acid position 17–36 comprises a major cross-neutralizing epitope and thus represents an attractive candidate antigen for broadly protective vaccination [6] – [8] . However, the major challenge in using the L2 protein as vaccine antigen is its rather poor immunogenicity compared to L1 VLP. It has been attempted to increase the immunogenicity of L2, by the use of different scaffolds [3] , [6] , [9] , linking of L2 to TLR agonists [10] or by using a concatenated L2 N-terminal fragment [11] .
Adeno-associated virus (AAV) belongs to the parvovirus family harboring a single-stranded DNA genome of approximately 4.7 kb. AAV particles that are very stable over a wide range of pH and temperature have a diameter of 25 nm, and are composed of 60 capsid protein subunits, designated VP1, VP2, and VP3 [12] . A recent study demonstrated that assembly of AAV capsids requires the expression of a virally encoded assembly-activating protein (AAP) [13] . Moreover, co-expression of VP3 with AAP allows the efficient production of virus-like particles (AAVLPs) composed of only the major capsid protein VP3 [13] . These capsids adopt an identical external capsid structure as VP1, VP2 and VP3 containing capsids [14] and represent a preferred scaffold for peptide vaccination, because of their simplicity and their inability to transfer genes which may inadvertently have been packaged in such AAVLPs. Due to the highly structured and repetitive presentation of epitopes on the capsid (60 times), combined with the intrinsic immunogenicity of AAV [15] , potent B-cell responses against the peptide presented at immunogenic capsid positions can be expected. So far, AAV capsids have not been analyzed for this purpose.
Here we show for the first time the potential to present B-cell epitopes on AAVLP capsid surface. We generated AAVLPs displaying HPV16 L2 epitopes in position 587 and HPV31 L2 epitopes in position 453. Our results demonstrate cross-neutralizing responses against several HPV types and therefore suggest AAVLP(HPV16/31L2) particles as a broadly protective vaccine candidate.
Adeno-associated virus serotype 2 (AAV2) capsids tolerate insertion of peptides in their antigenic region at position 587 or 453 [16] – [22] that are attractive sites for presenting B-cell epitopes.The expression of only two AAV proteins, VP3 and AAP, allows the formation of VP3-VLPs (AAVLPs). These observations suggest that recombinant AAVLPs could be generated with insertions of peptides in positions 587 and 453. After immunization with HPV16 L2 peptide 17–36 a broad range of cross- neutralizing antibodies was generated, but low or no antibodies against HPV31 [23] . Since HPV L2 17–36 aa sequences of HPV16 and 31 are clearly different, we incorporated both HPV L2 peptides into AAVLP particles at position 587 and 453 ( figure 1A). Stretches of 3 or 5 glycine residues were added to the N and C termini of the respective peptides for optimal surface display and several amino acids resulting from restriction enzyme sites used for cloning increased the size of inserted peptide sequences to 35 aa (position 453) and 31 aa (position 587). The amino acids at positions 585 and 588 were substituted. These R585A/R588A substitutions increased the titers of AAVLP particles with inserts. SDS-PAGE and Western blot analysis of AAVLPs confirmed the identity and size (approximately 66 kDa of AAVLP(HPV16/31L2) VP3) of the purified protein ( figure 1B). Presentation of the inserted HPV L2 peptides on the AAVLP particles were demonstrated with L2-specific rabbit sera in a dot blot assay under native conditions. However, since these sera cross-react with the HPV16 and HPV31 displayed peptides, exposure of both of them could not be unambiguously proven. As shown in figure 1C, proteins assembled into VLPs of ∼25–30 nm in diameter. In conclusion, the insertions of 35 aa (position 453) and 31 aa (position 587) into the capsid protein did not interfere with VLP assembly.
A) Schematic presentation of the chimeric VP3 protein with the insertions of the HPV31 and HPV16 L2 peptides. The amino acid residues refer to the VP1 protein sequence. Solid lines indicate the VP3 protein and open bars the L2 peptides. B) Analysis of the purified AAVLP(HPV16/31L2) particles by SDS- PAGE, coomassie blue staining and Western blotting with the monoclonal antibody B1 directed against VP3. Native dot blots with polyclonal rabbit sera to the HPV16 and 31 L2 aa 17–36 peptides, AAVLP particles and the AAVLP(HPV16/31L2) particles. Dot blots are done in duplets. C) TEM of the purified particles.
In order to analyse the HPV16 L2-specific immune response induced by AAVLP(HPV16/31L2) particles we immunized C57BL/6 mice either with a low dose (LD) (1E+11 particles equivalent to 0.6 µg of protein) or high dose (HD) (5E+12 particles equivalent to 30 µg) with or without adjuvant (+M) (montanide ISA 51). Thioredoxin-L2(20–38)
C57BL/6 mice (5 per group) vaccinated twice intramuscularly with AAVLP(HPV16/31L2) particles either with a low dose (LD) (1E+11 particles equivalent to 0.6 µg of protein) or high dose (HD) (5E+12 particles equivalent to 30 µg) with or without an adjuvant (+M) (montanide ISA 51). As positive control, mice were immunized three times subcutaneously with Thioredoxin-L2(20–38)
Neutralization was determined by inhibition of gene transduction by pseudovirions (PsV) containing the gene for gaussia luciferase (gLuc). Transduction was measured by gLuc expression which is taken as a surrogate of infection. The sera were tested in serial dilutions from 1∶40 to 1∶30,720, and a 50% reduction of gLuc activity was defined as positive neutralization. As shown in figure 3A, the enhancing effect of the adjuvant was also evident from the neutralizing activity against HPV16 and HPV31, but the HPV31-specific neutralization was weaker. Future HPV vaccines should induce immunity against other high-risk HPV types. We therefore analysed the ability of our particles to induce cross-neutralizing antibodies against several PsV (HPV18, HPV45, HPV52 and HPV58). As shown in figure 3B a similar pattern of reactivity was observed when the sera were tested for cross-neutralizing antibodies against HPV18, HPV45, HPV52 and HPV58. Animals vaccinated with TrXL2+M did not develop cross-neutralizing antibodies. We conclude from these results that neutralizing activity against HPV16 induced by the particles is higher than the one to HPV31. Cross-neutralization can only be achieved with adjuvanted AAVLPs. Reactivity of sera from mice immunized with AAVLP(HPV16/31L2) particles was higher than TrXL2 in C57BL/6 mice.
A) Neutralizing antibodies in sera of immunized animals were analyzed by HPV16 and HPV31 pseudovirion infection assay. B) Cross-neutralizing antibodies against HPV18, HPV45, HPV52 and HPV58 pseudovirions were detected by pseudovirion infection assay. Data of individual mice are shown. Data are expressed as the reciprocal neutralization titer obtained by using a cut off of 50% neutralization activity by incubation with serial dilutions from 1∶40 to 1∶30,720 of the sera. Mean titers of each group are shown by horizontal bars.
Cross-neutralizing activity of AAVLP(HPV16/31L2) was more closely analyzed in Balb/c mice, to this end animals were immunized with low dose of AAVLP plus montanide ISA51 two (LD+M 2x) or three times (LD+M 3x). In order to test the potency of another adjuvant a group of mice were vaccinated three times i.m. with low dose of AAVLP together with monophosphoryl Lipid A (MPL) (LD+MPL). Animals vaccinated with TrXL2 plus montanide ISA51 (TrXL2+ M) or with one of the two licensed HPV vaccines, Cervarix™ and Gardasil™ (1/10 of the dose recommended for humans) were used as controls.
A potential challenge for using AAV2-based vaccines in humans is the naturally acquired immune responses in approximately 80% of the human population ( [24] , [25] , [15] ), as the presence of antibodies against the AAV capsid may prevent an efficient vaccination. Therefore we immunized a group of animals with AAV2 (1E+11 particles/animal) prior to vaccination with AAVLP. Two and four weeks after pre-immunization, antibodies reacting with AAV2 capsids were detected by ELISA (titers between 80 and 1000) ( figure 4). At week 4 animals were vaccinated with LD+M 3x. Generation of HPV16 L2 specific antibodies was measured by a GST-L2 ELISA two weeks after the last immunization. As shown in figure 5, mice immunized with two or three doses of AAVLPs as well as with TrXL2+M developed comparable high titers of L2-specific antibodies (>100,000). Interestingly, also after pre-immunization with AAV similar titer levels were measured. The requirements for vaccines that are stable at ambient temperature imply the development of freeze-dried formulations may be beneficial. Lyophilized and rehydrated AAVLP(HPV16/31L2) particles were shown to be only slightly less immunogenic compared to the unprocessed particles. Animals vaccinated in the presence of MPL developed around one log lower HPV16 L2-specific antibody titers (<10,000) than mice which received montanide ISA51 adjuvanted particles.
Ten Balb/c mice were immunized twice intranasally with AAV2 (1E+11 capsids). Two (black bars) and four weeks (grey bars) after the first immunization sera was tested for detection of AAV-specific capsid antibodies using an AAV-based ELISA. Data are expressed as reciprocal titers of individual animal.
Mice were immunized with low dose of AAVLP(HPV16/31L2) particles either twice (LD+M 2x) or three times (LD+M 3x) i.m. together with montanide ISA51 as an adjuvant. A group of animals were vaccinated three times intramuscularly (i.m.) with low dose of AAVLP(HPV16/31L2) particles together with monophosphoryl Lipid A (MPL) as an adjuvant (LD+MPL). In addition, one group of mice was immunized with three low doses of the lyophilized AAVLP(HPV16/31L2) particles in combination with montanide ISA51 (Lyophilized). Four weeks after vaccination with AAV2 a group of animals were i.m. vaccinated with AAVLP(HPV16/31L2) three times with montanide ISA51 (AAV preimmune). Animals vaccinated 3 times subcutaneously (s.c.) with thioredoxin-L220–38 (50 µg) plus montanide ISA51 (TrXL2+ M) and a group of animals immunized with Cervarix™ and Gardasil™ were used as controls. Six weeks after the first immunization HPV16 L2-specific antibodies were detected by GST-L2 ELISA. Data of individual mice are shown. Mean titers of each group are shown by horizontal bars.
Neutralizing activity of the different groups against HPV16 and HPV31 PsV followed a similar pattern as the titers measured by ELISA with responses against HPV31 being consistently lower than against HPV16 ( figure 6A). Sera of all mice vaccinated with TrXL2+M, Cervarix™ and Gardasil™ had neutralizing activity against HPV16 PsV with mean titers of >10000 but not HPV31 PsV. Cross-neutralizing antibodies against HPV18, HPV45, HPV52, HPV58 and BPV PsV were detected, again in a similar ratio between the groups with neutralizing activity being highest against HPV18, HPV45 and HPV58 ( figure 6B). Most of the animals immunized with TrXL2+M developed HPV45, HPV52, HPV58 and BPV cross-neutralizing antibodies. Low level of cross-reactivity against HPV45 was obtained with 2/5 animals that had received Cervarix™.
A) Neutralizing antibodies in sera of immunized Balb/c animals were analyzed by HPV16 and HPV31 pseudovirion infection assay. B) Cross-neutralizing antibodies against HPV18, HPV45, HPV52, HPV58 and BPV pseudovirions were detected by pseudovirion infection assay. Data of individual mice are shown. Data are expressed as the reciprocal neutralization titer obtained by using a cut off of 50% neutralization activity by incubation with serial dilutions of the sera. Mean titers of each group are shown by horizontal bars.
In conclusion, Balb/c and C57BL/6 mice responded with similar HPV16- specific titers to immunization with AAVLP(HPV16/31L2) particles, but the cross-neutralizing titers were higher in Balb/c mice. Remarkably, pre-existing AAV antibodies did not prevent the induction of humoral responses against HPV. Lyophilized particles retained immunogenicity after reconstitution, albeit at somewhat lower level. In both mouse strains immune responses against HPV16 induced by AAVLP is higher than against HPV31, suggesting that the HPV31 L2-epitope inserted at position 453 is less immunogenic than the HPV16 L2-epitope inserted at aa 587.
In addition, three ZIKA hybrid rabbits were immunized four times with AAVLP(HPV16/31L2) particles (2E+12 capsids equivalent to 13.2 µg) adjuvanted with montanide ISA720. When analyzed two weeks after the last immunization, all animals developed HPV16/31 L2-specific antibodies with titers around 100,000 ( figure 7A). The antibodies neutralized PsV of HPV16 and HPV31 at dilutions higher than 1∶1000 ( figure 7B). Cross-neutralizing titer against HPV18 was similar, whereas cross-neutralization of HPV45, HPV52 and HPV58 PsV was lower but still significant. These data support the high and broad reacting immune response induced by AAVLP(HPV16/31L2) particles also in this animal model.
Three rabbits were immunized i.m. four times with AAVLP(HPV16/31L2) particles (2E+12 capsids equivalent to 13.2 µg) together with montanide ISA720 as an adjuvant. A) Ten weeks after the first immunization sera was analyzed by an L2 peptide-based ELISA to detect HPV16 and HPV31 L2-specific antibodies. B) The presence of neutralizing and cross-neutralizing antibodies against six different HPV types (HPV16, 18, 31, 45, 52, 58) was measured by pseudovirion-based neutralization assay (PBNA). C) Naïve Balb/c mice were injected i.p. with 100 µl buffer, 20 µg RG-1 purified monoclonal antibody or rabbit antiserum to AAVLP TP18 or serum from three different rabbits vaccinated with AAVLP(HPV16/31L2). One day later, the mice were vaginally challenged with HPV16 pseudovirus carrying a luciferase reporter. At three days post challenge, infection was assessed by imaging luciferase activity.
To test whether the rabbit sera raised against AAVLP(HPV16/31L2) confer protection against HPV16 infection, in an in vivo model, rabbit sera were administered intraperitoneally to naïve mice. Rabbit sera against AAVLP with an epitope of the cholesteryl ester transfer protein (AAVLP TP18) were used to detect the unspecific effect of AAVLP induced antibodies. Vaginal infection of mice is detected three days after challenge as luminescence signal after injection of the challenged mice with luciferin. The passive transfer of AAVLP(HPV16/31L2) sera protected mice from vaginal challenge with HPV16 PsV, whereas the AAVLP TP18 control serum failed to protect the mice ( figure 7C). Protection by AAVLP(HPV16/31L2) sera was similar to RG-1, a neutralizing monoclonal antibody recognizing the L2 17–36 motif.
The first steps in papillomavirus infection involve L1 dependent binding of the viral capsid to heparan sulfate proteoglycan [26] and lamin 5 in the extracellular matrix [27] which induces a conformational change leading to the exposure of L2, furin dependent proteolytic processing and attachment to keratinocytes [28] , [29] . Intervention with this infection pathway by prophylactic vaccination has two possible targets, the L1 or the L2 protein of the HPV capsid. While L1 antibodies are able to prevent both steps – attachment to basal membrane and to keratinocytes – L2 antibodies can interfere only with binding to the keratinocytes. L1 antibodies can be induced to high neutralization titers, especially when generated by HPV-VLPs, however, they have the drawback of HPV-type restriction. Furthermore, L1 VLPs are expensive to produce, especially for highly multivalent vaccines, and distribution of such vaccines requires a cold chain which limits their adoption by developing countries. Thus, although two approved L1 HPV-VLP vaccines – Gardasil™ and Cervarix™ – are highly protective against the main high-risk HPV infections, they lack cross-protection against other, more rare high-risk HPV types, they are not heat stable and are expensive to produce. L2 vaccines on the other hand, are able to expand the cross-protection profile due to extensive homology of the N terminus of L2 [30] . In particular, oligopeptides of the L2 proteins are sufficient to induce a broadly cross-neutralizing immune response [31] . However, neutralizing titers generated by L2 peptides are relatively low compared to those produced by L1VLPs [32] .
Several attempts have been made to improve the immunogenicity of L2. These include insertion of L2-neutralizing epitopes on the surface of HPV VLPs [9] , [33] , construction of tandem repeats of one peptide on bacterial thioredoxin [3] or concatenated L2 peptides from different papillomavirus types [11] . We have chosen AAVLPs as a platform for presentation of a cross-neutralizing L2 epitope (aa 17–36). AAV capsids tolerate insertion of peptides at different positions of the capsid and can be produced in large quantities from only one capsid protein making it a simple, non-infectious, empty capsid vaccine molecule [13] , [18] , [19] , [20] . In this first exploration of AAVLPs as a vaccine for generating a humoral immune response, the anti-peptide titers obtained were remarkably high (in the range of 10 4 to 10 5) after two immunizations with only 0.6 µg VLPs per dose. A third immunization only slightly increased the anti-L2 peptide titers. Nevertheless, an adjuvant seems to be required for AAVLP vaccination, because the recombinant AAV capsid alone elicits only a weak or no innate immune response. So far, only one alternative adjuvant (MPL) has been tested which was not as effective as montanide. Other options have still to be explored [34] , [35] . Pre-existence of anti AAV antibodies which are highly prevalent in the human populations, rather boosts than prevents the generation of the L2 specific immune response. This could be explained by improved, anti-AAV antibody Fc-mediated uptake of AAVLPs by professional antibody presenting cells. A particular advantage of AAVLPs is their stability which allows lyophilization and rehydration [36] . Thus, lyophilization of AAVLPs likely permits storage and delivery of AAVLPs under harsh conditions and in principle circumvents the need of a cold chain for vaccine distribution. This may be an important cost factor in developing countries and should be further explored.
The AAVLPs of this study can be produced by transient transfection of one or two plasmids expressing the capsid protein VP3 and the assembly activating protein AAP [13] . Under these conditions production of 10 12 to 10 13 capsids/ml can be achieved. Purification of the particles follows standard chromatography procedures and does not constitute a bottle-neck for AAVLP manufacturing. Co-expression of VP3 and AAP using baculovirus based vectors in insect cells provides one possibility of scale-up for clinical applications ( [37] , our own results). Methods for less costly AAVLP production, e.g. using bacteria or yeast, have still to be explored.
Immunization with AAVLPs containing the insertion of two different L2 peptide sequences at two separate positions of the same capsid provided a broadly cross-neutralizing antiserum, as tested in a pseudovirion (PsV) infection assay. This was observed in C57BL/6 mice, Balb/c mice and in rabbits. The HPV16-L2 peptide inserted at 587 provided nearly 100% protection against HPV16 PsV infection with rather high neutralizing antibody titers. This was not the case for the HPV31-L2 peptide inserted at aa position 453 and tested in the HPV31 PsV-based neutralization assay. The result is in agreement with the study of Jagu et al. that showed that a multitype L2 fusion protein which included a peptide of HPV31 induced robust antibody titers against various HPV types, but had relatively low titers against HPV31 [11] . However, also the position of peptide insertion in the AAV capsid may play a role. Peptides at position 453 are more exposed on the AAV capsid surface and have a larger interpeptide distance at the threefold symmetry axis of AAV2 than peptides inserted at aa 587. Immunization with the same peptides inserted at the reversed positions should show whether the inherent immunogenicity of the two peptides or the localization on the capsid surface is responsible for the difference in the immune reaction. Such an analysis might provide valuable information for the optimal spatial arrangement of antigenic epitopes on viral scaffolds for vaccination.
Because vaccination at low dose applies only 0.6 µg of AAVLPs/dose, a number of combinations of different AAVLPs can be tested for optimization of the vaccine. This includes also combinations of different AAVLPs with insertions of different HPV-L2 peptides at one position or combined at two positions. Another option would be the insertion of L2 peptides into other immunogenic sites of the AAV capsid [38] . Furthermore, the combination of peptide insertions with the expression of antigens by gene transfer may provide a basis for a combined prophylactic and therapeutic vaccine.
Cross-neutralizing vaccines against HPV types may not only be prophylactic for cervical cancer and genital warts, but may also have an impact on the prevention of a subset of head and neck or skin cancers for which a causal role of certain HPV types has been described. In general, this study describes the potential of AAVLPs as a vaccination option for prevention of infection associated diseases upon insertion of the appropriate linear protective epitope.
All animal procedures were performed according to approved protocols and in strict accordance with institutional and national guidelines; Federal law and the standard ethical guidelines (NIH, 1985; European Communities Directives, 1986 86/609/EEC) and approved by local government authorities (Regierungspräsidium Karlsruhe, Germany), guide for the Care and Use of Laboratory Animals of the National Institutes of Health, with the prior approval of the Animal Care and Use Committee of Johns Hopkins University. All immunizations were performed under ketamine anesthesia as describe below, and all efforts were made to minimize suffering.
Human embryonic kidney (HEK) 293T cells [39] , and 293TT cells [40] were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal-calf serum, 100 U of penicillin/ml, and 100 µg of streptomycin/ml at 37°C in 5% CO
C57BL/6 ( H-2b) and Balb/c female mice at 6–8 weeks of age were purchased from Charles River Wiga (Sulzfeld, Germany) and kept in an isolator at the animal facilities of the DKFZ. ZIKA hybrid rabbits were purchased from Dr. Zimmermann GbR (Abtsgmünd- Untergröningen, Germany) and housed at the company BioGenes (Berlin, Germany). For HPV challenging female Balb/c (6–8 weeks old) were purchased from the National Cancer Institute (Frederick, MD, USA) and kept in the animal facility of the Johns Hopkins School of Medicine (Baltimore, MD, USA).
AAVLP particles were generated from a plasmid containing the overlapping AAV2 VP2 and VP3 coding sequence cloned into the XhoI and NotI site of the pCI plasmid (Promega, Madison, WI). The start codon of VP2 was destroyed by introducing a point-mutation using the Quick Change Site-Directed Mutagenesis kit (Agilent Technologies, La Jolla, CA). A FseI site was introduced without changing the VP3 protein sequence at position 520 (amino acid number relative to the VP1 protein of AAV2) to generate the plasmid pCIVP2mut. In order to introduce peptides into VP3 the plasmid pCIVP2mut was modified. The plasmid pCIVP2mut-I587 was generated by introduction of NotI and BspEI sites at position 587. A second plasmid, pCIVP2mut-I453, was generated by introduction of NotI and BspEI sites at position 453. The nucleotide sequence of L2 residues 17 to 36 of HPV16 was cloned into the NotI/BspEI digested pCIVP2mut-I587 and the nucleotide sequence of L2 residues 17 to 36 of HPV31 into the NotI/BspEI digested pCIVP2mut-I453. Subsequently a fragment containing the HPV31 L2 sequence was generated by digestion of pCIVP2mut-I453 with BsiWI/ FseI. The fragment was subcloned into the BsiWI/ FseI digested pCIVP2mut-I587 and finally arginines at position 585 and 587 were substituted by alanine residues to generate the plasmid for the AAVLP(HPV16/31L2) production.
293T cells were transfected with AAVLP(HPV16/31L2) plasmid DNA (36 µg per 15 cm dish). Transfected cells were harvested after 3 days and lysed by three freeze-thaw cycles. The medium was cleared by filtration and adjusted to pH to 6.0 in HEPES buffer. Particles were further purified through chromatography. Briefly, the diluted cleared lysate containing the AAVLP particles was loaded onto Fractogel EMD SO
Expression of the AAVLP(HPV16/31L2) VP3 proteins was verified by Western blotting using the monoclonal antibody B1 (diluted 1∶200, Progen). The blotted membrane was incubated with 5% skim milk in 1 x PBS/0.05% Tween-20 for 1 h at RT followed by incubation of the membrane with antibody B1 for 1 hour at RT. After washing bound antibodies were detected with 1∶2,500 diluted HRP-labeled anti-mouse IgG (DAKO, Glostrup, Denmark). The expression of the AAVLP HPV16 and HPV31 L2 inserted peptides was analyzed by native dot blotting. Two µg (3E+11 particles) of the AAVLP(HPV16/31L2) particles, 2 µg of AAVLP particles without inserts or 10 µg of the HPV16 and 31 L2 peptides QLYKTCKQAGTCPPDIIPKV and QLYQTCKAAGTCPSDVIPKI were dotted on a nitrocellulose membrane that was incubated with 1∶1,000 diluted rabbit sera for 1 hour at RT. Bound antibodies were detected with 1∶2,500 diluted HRP-labeled anti-rabbit IgG (DAKO, Glostrup, Denmark).
Lyophilization of particles in 600 mM mannitol dissolved in Hepes buffer pH 6.0 with 200 mM NaCl was carried out using Epsilon 2-12D freeze-drier (Martin Christ Freeze Dryers GmbH, Osterode, Germany) stored at ≤20°C until use.
Transmission electron microscopy (TEM) of the purified AAVLP(HPV16/31L2) particles was performed after fixation 2.5% glutardialdehyde and stained with 2% uranium acetate and 0.01% glucose (performed by Prof. Dr. Wanner at the LMU Munich, Germany).
Particles were produced as described previously in [36] . Briefly, 293T cells were infected with a recombinant adenovirus expressing VP1, VP2 and VP3 of AAV2. Cells were harvested and fractionated on a sucrose cushion by under laying the supernatant with 450 µl of 30% sucrose (in Tris-EDTA) followed by 450 µl of 50% sucrose in Tris-EDTA. The gradients were centrifuged at 273,000× g for 2.5 h at 4°C by using an SW60 rotor (Beckman, Germany). The pellet was resuspended in TBSM buffer and purified on a second sucrose cushion. The resulting pellet was resuspended in an appropriate volume of TBSM buffer and then analyzed by electron microscopy.
Female mice were kept under deep ketamine 10%/rompun 2% (Bayer) anesthesia by intraperitoneal injection. Low doses (1E+11 particles/dose) or High doses (5E+12 particles/dose) of AAVLP(HPV16/31L2) were delivered intramuscularly into the tibia anterior muscle of the right leg with or without adjuvant. Two weeks later a boost was administrated in the same way. In case of TrX-L2 immunization, 50 µg of filter-sterilized TrX-L2(20–38)
Montanide ISA51 and montanide ISA720 were obtained from Seppic (Paris, France). A mixture of 1∶1 (ISA51) or 1∶2 (ISA720) with the antigen was applied per dose. Five micrograms of monophosphoryl lipid A derived from Salmonella enterica (MPL) in 2% squalene oil-in-water emulsion (Sigma Adjuvant System, Sigma, St. Louis, MA) were applied per dose.
The presence of HPV16 L2-specific IgG antibodies in sera of immunized mice was determined by GST L2-ELISA as described [3] . To detect anti-AAV antibodies sera (in 2-fold dilutions starting from 1∶50 to 1∶500,000) were added into AAV2 pre-coated plates (10 10 particles/well). HRP-coupled antimouse IgG was used as secondary antibody (dil 1∶5000, Southern Biotechnology, Birmingham, MA). TMB (Sigma) substrate solution was used as substrate OD was measured in an ELISA reader at 450 nm. Nonspecific binding was determined by using the same dilutions on plates coated with PBS only. The presence of HPV16 and HPV31 L2-specific IgG antibodies in sera of immunized rabbits was determined by L2 peptide-ELISA. 96-well plates were coated with 1 µg/well of the peptides QLYKTCKQAGTCPPDIIPKV or QLYQTCKAAGTCPSDVIPKI in PBS overnight at 4°C and blocked with 5% skim milk in PBS-T for 1 h at 37°C. The peptides were incubated with serial dilutions of the sera (3-fold dilutions starting from 1∶1,000 to 1∶2,187,000) in PBS-T with 1% skim milk and 1% BSA for 1 h at 37°C. Rabbit pre- immune sera were diluted 1∶1,000. Bound rabbit IgGs were detected after incubation with a 1∶2,500 diluted HRP-labeled anti-rabbit IgG (DAKO) antibody following the same conditions as above.
Neutralizing antibodies in sera of immunized animals were determined as described previously [4] but with pseudovirions (PSV) expressing gaussia luciferase (gLuc) as reporter. Briefly, PsV were prepared by transfecting 293TT cells with a plasmid encoding for the humanized HPV16 L1 and L2 genes, together with a plasmid containing the gene for gLuc under the control of CMV promoter. For PsV extraction, 293TT cells were harvested and washed once with 1 ml of Dulbecco’s Phosphate Buffered Saline (DPBS; Gibco). The cell pellet was resuspended in 150 µl of lysis buffer (140 µl DBPS, 9 µl 10% Brij58 (w/v) (Sigma) and 1 µl RNAse A/T cocktail (Fermentas)). The PsV-containing supernatant was set aside and the cell pellet was resuspended anew in 300 µl DPBS/0.8 M NaCl. Samples were cleared by centrifugation at 10,000 rpm and 4°C for 10 minutes. The supernatants were combined, 2 µl benzonase were added and the samples were incubated for 1 hour at 37°C. The PsV were Optiprep purified and used for infection of HeLaT cells.
Sera were transferred to 384 well polypropylene V-bottom plates (Greiner) and serially diluted ten times in 2-fold increments from 1/3 to 1/1536. Each 2 µl of the serially diluted sera were transferred to 21 white 384 well cell culture plates (PerkinElmer) with an EP3 384-head pipetting robot (Perkin Elmer), the plates immediately sealed with a polypropylene cover foil and stored at −20°C.
HPV16 pseudovirions with encapsulated luciferase reporter were generated by cotransfection of 293TT cells with plasmids encoding codon-modified L1 and L2 and a firefly luciferase reporter plasmid, as described previously [40] . Female Balb/c mice were treated with 3 mg of Depo-provera (Pfizer) 4 days prior to viral challenge. One day prior to challenge, mice were injected i.p. with buffer, undiluted rabbit antisera or 20 µg of purified RG-1 monoclonal antibody. The mice were anesthetized and challenged with pseudovirus as previously described [42] , but with modifications. Briefly, a HPV16 PsV inoculum of 20 µl mixed with 20 µl of a 3% CMC preparation, based on L1 content, was used. The inoculum was delivered using an M20 positive-displacement pipette in two doses; 20 µl before and 20 µl after insertion in the vagina of a Cytobrush that was turned clockwise and counterclockwise 10 times. After the challenge, standard dissecting forceps were used to occlude the vaginal introitus to achieve maximal retention of the material while the mice recover from anesthesia. Three days after pseudovirion challenge, the mice were again anesthetized and 20 µl of luciferin (7.8 mg/ml) was deposited in the vaginal vault. Luciferase signals were acquired for 10 min with a Xenogen IVIS 100 imager, and analysis was performed with Living Image 2.0 software (Caliper Life Sciences). An identical region of interest (ROI) was drawn around the luciferase signal emitted from each mouse, and the average radiance within the ROI was determined.
We want to thank Prof. Dr. Gerhard Wanner of the faculty of biology at the LMU in Munich, Germany for making the electron microscopic pictures.
Competing Interests: The authors have read the journal’s policy and have the following conflicts: MW, MR, SS, MK, MH and UM are or were employees of MediGene AG when the study was performed. MR, SS, MK, MH and UM hold stocks or stock options of MediGene AG. The corresponding author had full access to all the data and had the final editorial rights of the manuscript. MR and MH are inventors to a patent application with the application number PCT/EP2011/004528 entitled “Cross-protective HPV L2 vaccine based on AAVLPs” filed by MediGene AG. RBSR is a co-inventor on L2 patents licensed to Shantha Biotechnics Ltd., GlaxoSmithKline, PaxVax, Inc. and Acambis, Inc. The terms of these arrangements are managed by Johns Hopkins University in accordance with its conflict of interest policies. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
Funding: RBSR and BK were funded by grants from the PHS (P50 CA098252 and RC2 CA148499), JAK and MM were supported by the Deutsche Krebshilfe, grant 109653. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


I'm a self-taught front-end web developer with experience working on backend code. I enjoy working on web applications, small projects and part of a team.
I've worked with startups, entreprenuers, non-profits to develop their websites and responsive mobile applications.
I am committed to life-long learning and look forward to working with you on your tech project.


Description
Message from your own Leaders
Jabalpur is a city in the state of Madhya Pradesh, India.
According to the 2011 census, it is the third-largest urban agglomeration in Madhya Pradesh and the country's 26th-largest urban agglomeration.
Mayor: Dr. Swati Godbole
Commissioner: Mr. Ved Prakash
CITIZENS MAY ALSO POST THEIR IDEAS ,SUGGESTIONS AND SOLUTIONS AT "MYGOV.IN".
Seven cities in Madhya Pradesh Bhopal, Indore, Gwalior, Jabalpur, Satna, Ujjain and Sagar will become Smart City. This important decision headed by the Prime Minister in New Delhi has been taken in a Cabinet meeting. It got decided in the meeting to create 100 smart cities across the country, including the four metropolitan cities of Madhya Pradesh. Where smart city will be rebuilt, what will be its nature is to be decided by the state government. Centre for Smart City Guide line of three is fixed. This involves redeveloping large colonies of these cities, re-densification by breaking old buildings and creating a smart city in green fields. State government committee on September 30 to 15 to create a framework for smart city centre will tell. December 15 by the City's plan to send him. According to instructions given by Central Government the Smart City must be formed within 5 years.
The strategic components of Area-based development in the Smart Cities Mission are city improvement (retrofitting), city renewal (redevelopment) and city extension (greenfield development) plus a Pan-city initiative in which Smart Solutions are applied covering larger parts of the city. Below are given the descriptions of the three models of Area-based Smart City Development:
The core infrastructure elements in a Smart City would include: adequate water supply, assured electricity supply, sanitation, including solid waste management, efficient urban mobility and public transport, affordable housing, especially for the poor, robust IT connectivity and digitalization, good governance, especially e-Governance and citizen participation, sustainable environment, safety and security of citizens, particularly women, children and the elderly, and health and education.
 The city is also blessed with holy river Narmada.
Have a glance at few of the sites.
The Dhuandhar Falls is the most beautiful waterfall of Madhya Pradesh, located on Narmada River in Bhedaghat. The plunges in waterfall creates a bouncing mass of mist due to smoke so called as Dhuandhar and very powerful roar which can be heard from a far distance.
Bhedaghat is a town famous for its bathing ghat of sacred river Narmada,Its another major attraction is Bandar Kodini and travels in between the marble rocks in a boat. In a moonlit night the marble rock mountains alongside of the river Narmada is the most beautiful site which should not be missed for anything.
The Chausat Yogini Temple of Jabalpur is one of two famous Chausat Yogini Temple in Madhya Pradesh, Chausath Yogini Temple Khajuraho being other. 64 Yogini temple is one of the four major extant temples made of coarse granite and Lord Shiva temple is situated in the middle of complex.
The most beautiful ghat of sacred Narmada river and famous for its temples such as Shri Ramlala Mandir, Ganesh Mandir,Gurdwara and Jain temple. Gwarighat has a Maa Narmada temple near to the bank but half of the temple always drown in water and Narmada temple aarti is should not be missed in the evening.
The Gurdwara Gwari Ghat Saheb is situated in the bank of holy river Narmada and that ghat is known as Gwari Ghat, Gurdwara is surrounded with a very picturesque locations. Complex of Gurdwara Gwarighat includes Guru ka Langar, pilgrims rooms,small museum and Homoeopathic dispensary.
The area of Madan Mahal is famous for the historical Durgavati fort Situated atop a hill in Jabalpur Madan Mahal Fort is a popular ancient fort of Jabalpur city and ranked as one of the top 10 among attractions in Jabalpur city.
Balancing rock of Jabalpur is an independent rock balancing over another rock, It is one of the two big balancing rocks in India, balancing rock mahabalipuram being other.
The Lord Shiva statue at Kachnar City in Jabalpur is 76 feet tall and one of the tallest statues of lord Shiva in sitting posture in India. Kachnar city Lord Shiva statue is situated under open sky and offers beautiful view of The Destroyer, also one of the tallest statues in India.
Hanumantal Bada Jain Mandir is one of the most famous and historic Jain temple, situated right on the edge of Hanumantal. The Hanumantal Jain Mandir is the largest independent Jain temple in India and main Jain temple in Jabalpur Pisanhari Ki Madiya is a another most famous Jain pilgrimage located near the Netaji Subhash Chandra Bose Medical College in Jabalpur.
Jabalpur Municipal Corporation
<EMAIL_ADDRESS>+91<PHONE_NUMBER>
For more information on Smart Cities and Challenges,
Check out- Smart City


Available for full-time positions or short-term work
Please refer to my website and resume below for more details
Khizer Asad is a recent graduate looking for a professional or research position. He is keen on pursuing a career in the public sector or with an NGO, with the goal of serving those most in need. His research interests include the intersection between public policy and data science so to pursue evidence based policies.
Current work includes research on political and social sentiments in Pakistan using data from Twitter’s API, using machine learning and NLP strategies. In addition to this, he is working on independent programming and statistics projects (read more in blog section). Khizer is very passionate about E-Sports, and in his free time freelances as a coach and commentator.
Download my resumé.
MicroMasters in Data, Economics, and Development Policy
MIT
BSc in Political Science, Statistics, and Mathematics
University of Toronto
More information available on my LinkedIn
TBA soon!


Today's DiscussionThe National Weather Service Forecasts-
TODAY...RAIN. HIGHS AROUND 49. SOUTHEAST WIND 15 TO 25 MPH
DECREASING TO 5 TO 15 MPH IN THE AFTERNOON.
TONIGHT...MOSTLY CLOUDY. AREAS OF FOG. CHANCE OF RAIN. LOWS 36 TO 42. EAST WIND 10 MPH.
FRIDAY...DECREASING CLOUDS. AREAS OF FOG IN THE MORNING. HIGHS AROUND 43. SOUTHEAST WIND 10 MPH SHIFTING TO THE NORTHWEST IN THE AFTERNOON.
Temperatures continue to remain quite warm. Temperatures have remained essentially above freezing at the Mt Roberts Tram Summit since 1/13/14. Temps have dipped below freezing but only for a few hours here and there.
The Eaglecrest Summit has been above freezing 5 out of the last 8 days.
Since the temps warmed on the 13th we have received 8.6" of rain at the Mt Roberts Tram Summit and the drainage channels are now well established in the snowpack.
We have lost nearly 80cm of snow since that time at the Tram Summit Wx Station.
Small wet avalanches remain possible but should not be large enough to pose a threat to the urban enviroment.
Glade rates have accelorated and we have seen a number of glide avalanches in the region. Be increasingly cautious is you choose to play in terrain that has evidence of snowpack gliding above it. Look for open glide cracks to give you a better idea of areas to avoid. These avalanches are very hard to predict yet be aware that during these continued warm temps danger is lingering in those areas.
Today's Avalanche TipRecognize and Survive Wet Snow Avalanche Conditions.
Two primary types of avalanches are slab avalanches and loose-snow avalanches, which begin at a point source and can occur with wet or dry snow conditions. Wet loose-snow avalanches, or wet avalanches, are encountered more frequently in spring and summer conditions as temperatures warm up and saturate the snowpack with water.
Wet avalanches can move slowly down a slope from its trigger point, usually spreading out in an inverted V configuration. But wet avalanches become even more dangerous when they load a snow-covered slope and then cause its underlying slab to give way. For this reason, a wet avalanche may lead to a larger and more dangerous slab avalanche.
Recognize Wet Snow Avalanche Conditions
As with other types of avalanches, knowledge of the Avalanche Triangle is important in helping you to evaluate avalanche danger. The Avalanche Triangle includes the key factors leading to avalanches: snowpack, weather, terrain, and human interaction.
Whereas dry snow avalanches occur when additional stress is put on the snowpack, wet avalanches occur as the snowpack strength decreases. Wet snow avalanche conditions develop when the snowpack is loose and saturated or moist with water. Water keeps the snowpack from consolidating in a stable manner; instead, snow becomes loosely associated and has a higher chance of sliding.
Spring weather contributes to wet avalanche conditions, especially when a storm causes rain instead of snow to fall on snow slopes. Rain adds weight to the snowpack, and it can also warm up the snow, causing additional melting. Rain can also saturate the snow, lubricating the layers, and making it easier for an avalanche to occur.
Wind is one of the most important weather factors to consider when evaluating dry avalanche danger since wind causes snowdrift and snow loading. But rain, melting caused by prolonged sun exposure, and warm temperatures are the more important weather factors to consider when evaluating wet avalanche potential.
Terrain variables including slope steepness, slope configuration, and slope aspect are important to consider when evaluating avalanche hazards, in general, but also consider how these variables contribute specifically to wet avalanche hazards.
Slope aspect, or the direction a slope faces, is an important factor to consider in spring or summer conditions. South-facing slopes receives more sun; therefore, south-facing slopes will be more prone to wet snow avalanches even though they are often considered to be more stable in the winter. Slope aspect considerations change significantly from winter to spring, so check local avalanche forecasts carefully.
Survive a Wet Snow Avalanche
Wet avalanches are most often triggered naturally, and they are more difficult for humans to trigger. In contrast, most of dry avalanches are caused by the avalanche victim or someone in the victim’s party, with some statistics citing human involvement in 90 percent of dry avalanche cases.
Prepare for wet avalanche conditions as you would prepare for other avalanche conditions by carrying essential avalanche gear including a beacon, a shovel, and a probe. As you are traveling into an area with avalanche potential, look carefully for recent wet avalanches. Often, if you see one, you’re in an area where conditions are right, and you’re likely to see more.
In wet snow conditions, an avalanche may slide down on victims traveling below it, making it seem as if victims are simply in the wrong place at the wrong time. For this reason, it’s even more important to consider terrain features and terrain traps when deciding where to travel.
If you are the victim of a wet snow avalanche, you are more likely to die from being crushed in the process of the avalanche, or you will suffocate quickly if you are buried. However, people are caught in wet avalanches less frequently than in dry avalanches.
As the victim, follow other avalanche survival advice, and do the following:
•Make sure that you and all others in your group are carrying essential avalanche gear.
•Shout in order to alert others of avalanche danger and of your location.
•Fight to stay on top of the avalanche if it begins to carry you away.
•Use other gear such as an Avalung™ or an avalanche airbag if you are swept away in an avalanche or if you see an avalanche slide coming your way.
•Create an air pocket as the avalanche slows if you are buried.
•Raise an arm or ski pole to mark your presence as an avalanche slows if you are buried close enough to the surface.
•Shout out to rescuers if you hear them; otherwise, remain calm while waiting for rescue if you are buried to conserve energy and oxygen.
|


Generating population projections for all countries of the world using several probabilistic components, such as total fertility rate and life expectancy.
| Version: |
7.0-0 |
| Depends: |
R (≥ 2.14.2), bayesTFR (≥ 6.0-0), bayesLife (≥ 3.0-1) |
| Imports: |
parallel, abind, plyr, wpp2017, wpp2012, graphics, grDevices, stats, utils, rworldmap, fields, googleVis, reshape2 |
| Suggests: |
wpp2015, wpp2010 |
| Published: |
2018-10-12 |
| Author: |
Hana Sevcikova, Adrian Raftery, Thomas Buettner |
| Maintainer: |
Hana Sevcikova <hanas at uw.edu> |
| License: |
GPL-2 | GPL-3 [expanded from: GPL (≥ 2)] |
| URL: |
https://bayespop.csss.washington.edu |
| NeedsCompilation: |
yes |
| Citation: |
bayesPop citation info |
| Materials: |
ChangeLog |
| CRAN checks: |
bayesPop results |


Welcome to About me page!
I love:
Pizza
Pizza
Pizza
and also, Pizza!
-------------------------------------
but Why I love Pizza?
It's healthy!
It's good for you!
It's pretty!
It doesn't have any drama!
It's so usefull!


Contact Us
About us
We are an organization of 225 hockey referees serving London and area. If you are currently looking for an organization to referee your hockey league, our team would like the opportunity to assist
About us
Our game starts here
The London referees Group is an organization of 225 hockey referees serving London and area. If you are currently looking for an organization to referee your minor hockey league or your mens hockey league.
Our History
Historic LRG Highlights
The name of the organization is the "London Referees' Group" and maybe referred to in the organization's documents and records as "LRG". LRG is an unincorporated private association of its members, established on 15 July 2007 at London, Ontanrio.
{{currentPeopleData.name}}
Role: {{currentPeopleData.role}}
Email: {{currentPeopleData.email}}
Click the image to see more infomation!


Far far away, behind the word mountains, far from the countries Vokalia and Consonantia, there live the blind texts. Separated they live in Bookmarksgrove right at the coast of the Semantics, a large language ocean.
A small river named Duden flows by their place and supplies it with the necessary regelialia. It is a paradisematic country, in which roasted parts of sentences fly into your mouth.
When she reached the first hills of the Italic Mountains, she had a last view back on the skyline of her hometown Bookmarksgrove, the headline of Alphabet Village and the subline of her own road, the Line Lane.
Even the all-powerful Pointing has no control about the blind texts it is an almost unorthographic life One day however a small line of blind text by the name of Lorem Ipsum decided to leave for the far World of Grammar.


Dreamhomes24X7 is an application for House Price prediction, sell, rent, buy and analysis. Web Development and Data Science have been a long time passion of ours. We took it as a challenge to integrate them using a new technology like flask. It has always been an idea to combine these two interests and merge them together and make a project. Finally, we have managed to accomplish this task by building a web app that predicts the real estate price for properties and houses across India. Our application is now live on the internet accessible from any place in the world.
Predict PriceWe help predict the estimated property price by selecting attributes.
We use Machine Learning algorithms and link them using flask to predict price easily on a web app.


--- layout: default title: Media permalink: /media/ ---
Some videos, blog posts, and other media I've been featured in.
{{medium.blurb}}


The paper shows that Siamese networks can be used for unsupervised learning with images without needing techniques like negative sample pairs, large batch training, or momentum encoders. The training mechanism is referred to as the SimSiam method.
Given an input image x, create two augmented views x1 and x2.
These views are processed by an encoder network f.
One of the views (say x1) is processed by the encoder f as well as a predictor MLP h to obtain a projection p1 ie p1 = h(f(x1)).
The second view (x2) is processed only by the encoder f to obtain an encoding z2 i.e., z2 = f(x2).
Negative cosine similarity is minimized between p1 and z2 with the catch that the resulting gradients are not used to update the encoder via z2. I.e., Loss = D(p1, stopgrad(z2)) where D is the negative cosine similarity and stopgrad is an operation that stops the flow of gradients.
In practice, both p1, z2 and p2, z1 pairs are used for computing the loss. ie Loss = 0.5 * (D(p1, stopgrad(z2)) + D(p2, stopgrad(z1))).
Encoder uses batch norm in all the layers (including output) while projection MLP uses batch norm only in the hidden layers.
SGD optimizer with learning rate as 0.05 * batchsize / 256, cosine learning rate decay schedule and SGD momentum = 0.9.
Unsupervised pretraining on the ImageNet dataset followed by training a supervised linear classifier on the frozen representations.
Stop-gradient operation is necessary to avoid a degenerate solution. Without stop-gradient, the model maps all inputs to a constant z.
If the projection layer is removed, the method does not work (because of the loss’s symmetric nature). If the loss is also made asymmetric, the method still does not work without the projection layer. However, asymmetric loss + projection layer works.
Keeping the projection layer fixed (i.e., not updating during training) avoids collapse but leads to poor validation performance.
Training the projection layer with a constant learning rate works better in practice, likely because the projection layer needs to keep adapting before the encoder layer is sufficiently trained.
The method works well across different batch sizes.
Removing batch norm layers from all the layers in all the networks does not lead to collapse, though the model’s performance degrades on the validation dataset. Adding batch norm to the hidden layers alone is sufficient.
Adding batch norm to the encoder’s output further improves the performance but adding batch norm to all the layers of all the networks makes the training unstable, with the loss oscillating.
Overall, while batch norm helps to improve performance, it is not sufficient to avoid collapse.
The setup does not collapse when the cross-entropy loss replaces the cosine loss.
Given that the stop-gradient operation seems to be the critical ingredient for avoiding collapse, the paper hypothesizes that SimSiam is solving a different optimization problem.
The hypothesis is that SimSiam is implementing an Expectation-Maximisation (EM) algorithm with two sets of variables and two underlying sub-problems.
The paper performs several experiments to test this hypothesis. For example, they consider k SGD steps for the first problem before performing an update for the second problem, showing that the alternating optimization is a valid formulation, of which SimSiam is a particular case.
SimSiam achieves the highest accuracy among SimCLR, MoCo, BYOL, and SwAV for training under 100 epochs. However, it lags behind other methods when trained longer.
SimSiam’s representations are transferable beyond the ImageNet tasks.
Adding projection layer and stop-gradient operator to SimCLR does not improve its performance.


Reports class with too many constructors. Classes with
too many constructors are prone to initialization errors, and may often be better modeled as
multiple subclasses.
Use the field below to specify the maximum number of constructors a class is allowed to have.
Powered by InspectionGadgets


Author: Alex
Ingredients:
1 and 2/3 cups (210g) sifted all-purpose flour,* (spoon & leveled)
1/2 teaspoon baking powder
1/4 teaspoon baking soda
1/2 teaspoon salt
1/2 cup (115g) unsalted butter, melted
3/4 cup (150g) packed light brown sugar
1/4 cup (50g) granulated sugar
1 large egg, at room temperature
1/4 cup (60g) yogurt*
1/2 cup (120ml) milk*
1/4 (60ml) cup pineapple juice (reserved from the canned pineapple or use fresh)
1 Tablespoon (15ml) pure vanilla extract
Cooking Instructions:
- Preheat oven to 350°F (177°C).
- Prepare topping first: Place 1/4 cup of melted butter into a 9-inch pie dish or 9×2 inch (deep dish) round cake pan. Make sure it is a deep pan. Sprinkle with 1/2 cup of brown sugar. Top with pineapple slices and arrange cherries between the pineapple slices. See my photo for a visual. Set aside.
- Prepare the cake: In a medium bowl, mix together flour, baking powder, baking soda, and salt. Set aside. In a large microwave-safe bowl, melt butter in the microwave. Whisk in the sugars vigorously getting out any brown sugar lumps – mixture will be gritty. Whisk in egg, yogurt, milk, pineapple juice, and vanilla extract until combined. Slowly mix in dry ingredients until no lumps remain. Batter will be thick.
- Pour/spoon batter into prepared pan. Make sure it’s not overflowing– this recipe makes enough batter for your deep dish pan. If you find there is too much batter, make a couple cupcakes (20 minutes bake time) on the side. Bake for at 40-48 minutes. Cover loosely with aluminum foil (loosely to avoid the top from sticking to the foil) at the 20 minute mark to avoid browning. Allow to cool for 10 minutes.
- Invert cake on top of a cake stand or a large serving plate. Slice and serve warm, room temperature, or even cold. Try serving with vanilla ice cream – it’s a delicious combination. Cover cake and store for up to 3 days in the refrigerator.
HINT: I wonder if you can turn this cake right-side up?


--- layout: post title: Loops In Ruby And Javascript category: Tech ---
One of the major differences between coding in Ruby and coding in Javascript is the way each language handles loops.
Loops are a common part of every programming language. They allow a program to repeat the same action a set number of times or until a specified condition is met.
There are many different ways to create loops in Ruby. You can use specified keywords or methods. One way to perform an action a set number of times is to use the .times method, for example if you want print out a sentence three times:
3.times {p “I love Ruby!”}
If you want to create a loop until a specific condition is met you can youse the while keyword. The following loop will continue until the variable i is greater than or equal to 3.:
Ruby also has a bunch of different methods to loop through a data structure. Some of the methods include .each, .map, and .inject. These methods make it simple to find the sum of an array of integers:
array = [1,3,5,6]
array.inject{ |sum, num| sum + num} => 15
JavaScript on the other hand only offers two keywords for creating loops. They are the keywords for and while. These two keywords are powerful enough for you to create all of the different kinds of loops that are available in the Ruby language, you just have to work a little bit harder as a programmer.
The while loop in JavaScript behaves much like the while loop in Ruby, however the syntax is a little more detailed:
var n = 1;
For loops are useful for looping a given number of iterations, much like the .times method in Ruby. For loops take in 3 parameters: the first parameter defines a variable, the second parameter defines the number of iterations, and the third parameter increases or decreases the variable after each iteration.
If you want to loop through an Array in JavaScript you can use a modified version of the for loop called the for (x in y) syntax where x is a variable representing an increment able value and y represents an object with a length property such as an array. Here’s our previous Ruby example done in JavaScript:
That is a brief comparison of the differences of loops between Ruby and JavaScript. Hopefully you have a little bit better understanding after reading this post.
Thanks for reading.


BL Club to conduct workshops for B-School students
Live demo on commodity market will be a highlight
by Special CorrespondentThe Business Line Club, in association with Multi Commodity Exchange Investor Protection Fund, will organise workshops on commodity market for B-Schools at various locations across the country.
Of the total 50 such workshops, seven will be conducted in colleges in Vijayawada city from November 25. The venues include P.B. Siddhartha College of Arts and Science, Andhra Loyola Institute of Technology, SRK Engineering College, KL Business School, Gudlavalleru Engineering College, V.R. Siddhartha Engineering College, and P.V.P. Siddhartha Institute of Technology.
Experts from MCX will address students at the workshops planned to be organised in B-Schools and PG colleges of Andhra Pradesh, Tamil Nadu, Kerala, Pune and Mumbai.
The idea is to create a platform for students to learn the concept of commodity market in a more pragmatic way. Live demo of the commodity market interface will be a highlight. Commodity market expert Shrikant Kaundinya will be at the helm of these workshops in Vijayawada.


Friday night drama as deadline for January transfer window is announced
The January 2020 transfer window for Premier League clubs will close at 11pm on Friday, January 31, it has been announced.
The 2020 winter transfer period will open on New Year’s Day.
The timings were confirmed at the top flight’s November shareholders’ meeting in central London on Thursday, attended by chairmen and chief executives of the 20 clubs.
Any deadline-day deals will need to complete deal sheets between 9pm and 11pm on the 31st. Clubs will have until midnight UK time on the 31st to complete FIFA transfer matching system (TMS) information to apply for international clearance for new signings.
It promises to be a busier than usual January window with Manchester City ready to spend money on their defence and Tottenham perhaps looking to offload one or two stars nearing the end of their contracts.


After seeing that scrypt no longer works for windows:
I had to act, as I’m the maintainer of the scrypt package. The package can be found here https://bitbucket.org/mhallin/py-scrypt/. The original author gave me write access to the repository as he is no longer maintaining it.
scrypt is an important package and is used in several python libraries as dependency (e.g. python-bitshares,, steem-python, beem )
The reason why it failed unter windows, is that a library in the OpenSSL package is renamed in the newest windows version of it. By fixing the names, I could compile it again.
Compile scrypt in windows
A c compiler must be installed (e.g. Visual Studio 2017). Then,
Win64 OpenSSL v1.1.1a from https://slproweb.com/products/Win32OpenSSL.html must be installed to c:\\OpenSSL-Win64
A
1 | python setup.py build |
should now work.
Precompiled wheels
I uploaded also several pre-compiled wheels.
A
1 | pip install scrypt |
under windows should also work without having a compiler.
Anaconda
I pushed the changes to conda-forge and working packages are available for windows:
This page is synchronized from the post: ‘Fixing the windows build of the scrypt library’


const val SQL_CREATE: String
SQLite statement which creates the "children" table upon execution.
See Also
com.hexanovate.familytree.database.DatabaseHelper


@* * Copyright 2016 HM Revenue & Customs * * Licensed under the Apache License, Version 2.0 (the "License"); * you may not use this file except in compliance with the License. * You may obtain a copy of the License at * * http://www.apache.org/licenses/LICENSE-2.0 * * Unless required by applicable law or agreed to in writing, software * distributed under the License is distributed on an "AS IS" BASIS, * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied. * See the License for the specific language governing permissions and * limitations under the License. *@ @import iht.models._ @import iht.models.application.ProbateDetails @import iht.views.html._ @import uk.gov.hmrc.play.views.html._ @import iht.utils._ @import iht.views.html.application.overview._ @(ihtref: String, deceasedName: String, probateDetails: ProbateDetails)(implicit request:Request[_], messages: Messages) @iht_main_template_application(title = Messages("page.iht.application.overview.inreview.title"), browserTitle = Some(Messages("page.iht.application.overview.inreview.title")), isFullWidth=false, sidebarLinks= iht.views.html.application.status.post_submission_sidebar(ihtref, probateDetails, "page.iht.application.overview.inreview.sidebartitle") ) {


This course provides a comprehensive overview of conceptual and technical design topics related to dynamic mapping, topics typically considered under the cartographic research thrusts of Interactive Cartography and Geovisualization. Specifically, it discusses user interface (UI) and user experience (UX) design as applied for web maps, drawing from research and practice on Human-Computer Interaction, Information Visualization, Usability Engineering, and Visual Analytics, perspectives that students are unlikely to receive in other GIS courses. The course emphasizes mapmaking over map use (compared to 170) and the design of interfaces to maps rather than the maps themselves (compared to 370 and 572). The class is divided into two components: lectures and labs.
Check out past syllabi and finals projects below!
Instructor: Robert Roth
TA: Richard Donohue


A web framework (WF) or web application framework (WAF) is a software framework that is designed to support the development of web applications including web services, web resources, and web APIs. (wikipedia)
The World Wide Web (abbreviated WWW or the Web) is an information space where documents and other web resources are identified by Uniform Resource Locators (URLs), interlinked by hypertext links, and can be accessed via the Internet.
My name is Muhammad Hassan Khurshid, a human, born in Pakistan , I moved to Florence at age of 10 and grew up in this beautiful city from then.
Now I am working at Frigel as a developer.
Frigel is a multinational company, located in Florence that creates and sells industrial cooling systems.
More then a year
I used Genropy for three projects:
OneClick is a web platform that performs data analysis of energy and does a lot of other stuff.
But unfortunately I did not get the permission to show it publicly
How many people here know what Trello is?
Genropy + Trello = Genrello
gnrmkproject app -P base -a
Genrello is based on a really simple db model, that consists just of few tables:
class Table(object):
def config_db(self, pkg):
tbl = pkg.table('board', pkey='id', name_long='!!Board',
name_plural='!!Boards', caption_field='name')
self.sysFields(tbl)
tbl.column('name', name_short='Name',
validate_notnull=True, validate_case='c',
validate_notnull_error='!!Mandatory field')
tbl.column('description', name_long='!!Description')
tbl.column('position', dtype='I', name_long='!!Position')
tbl.column(
'team_id', size='22', group='_',
name_long='!!Team id'
).relation(
'team.id', relation_name='board_team',
mode='foreignkey', onDelete='cascade'
)
tbl.column(
'owner_user_id', size='22', group='_',
name_long='!!Owner'
).relation(
'adm.user.id', relation_name='board_user',
mode='foreignkey', onDelete='cascade'
)
tbl.aliasColumn(
'username',
<EMAIL_ADDRESS>name_long='!!Username'
)
tbl.aliasColumn('team_name',
<EMAIL_ADDRESS>name_long='!!Team name')
class Table(object):
def config_db(self, pkg):
tbl = pkg.table('board', pkey='id', name_long='!!Board',
name_plural='!!Boards', caption_field='name')
self.sysFields(tbl)
tbl.column('name', name_short='Name',
validate_notnull=True, validate_case='c',
validate_notnull_error='!!Mandatory field')
tbl.column(
'team_id', size='22', group='_',
name_long='!!Team id'
).relation(
'team.id', relation_name='board_team',
mode='foreignkey', onDelete='cascade'
)
tbl.aliasColumn('team_name',
<EMAIL_ADDRESS>name_long='!!Team name')
gnrdbsetup instanceName
gnrmkthresource projectName:packageName.tableName
Frametable is a core component of Genropy, that let you CRUD on the table and more!
def main_page(self, pane):
pane.textBox(value='^name', lbl='!!Name')
pane.dataRpc('welcome', self.myfunc, name='^name')
pane.div('^welcome')
@public_method
def myfunc(self, name):
return "Hello {}".format(name)
Genrello: genrello.mkshid.com
Genrello Source: https://github.com/mkshid/genrello
Mail<EMAIL_ADDRESS>Blog: http://www.mkshid.com
Twitter: @mkshid


WITNESS LIST
Redistricting Committee
January 9, 2020 - 4:00 PM
Interim Field Hearing on 2021 Redistricting
On:
Ackerman, Judy (Self)
Beechinor, Julia (Self)
Bell, Kenneth (Self)
Bretting, John (Self)
Dayoub, Richard (Self)
Galindo, Perla (Self)
Guida, Marilyn (Self)
Heyman, Robert (Self)
Hollandbyrd, Hannah (Hope Border Institute)
Ivey, James (Self)
Ivey, Jennifer (Self)
Ivey, Kevin (Self; West Texas Pecan Association)
Jones, Gil (Valley Gin Company)
May, Jared (Texas Legislative Council)
Powers, Karen (Self)
Reza, Ana (Self)
Stillman, Diana (Self)
Svarzbein, Peter (Self)
Swanson, Stephanie (Self)
Valdez, Jesus (Self)
Valencia, Lila (TX State Demographer)
Wallace, Carol (League of Women Voters of El Paso)
Weston, Carol (Self)
Wiebe, John (UTEP - Administration)
Wigle, Jean (Self)
1


rip
-- rip file format
A simple Rip file (e.g. deps.rip):
git://github.com/ezmobius/redis-rb.git eed200ad
git://github.com/brianmario/yajl-ruby.git 0.6.3
rack
sinatra
mustache
A more complex Rip file:
git://github.com/ezmobius/redis-rb.git @1.0 =>redis
git://github.com/brianmario/yajl-ruby.git 0.6.3
sinatra 0.9.4
* rack 1.0
rake # any version
!brew install mongodb
# Classier with caps
git://github.com/quirkey/vegas.git v0.1.2 =>Vegas
logging.rip
git://github.com/rails/rails.git /activerecord
Each Rip file describes a set of Rip packages, one package per line.
A basic Rip package can be described in terms of its source and relevant install-time instructions.
The format is:
source [install options]
Each line of a Rip file can be fed to rip-install
.
Rip files use Ruby-style #
comments. Anything after a #
on a line
is considered a comment.
# comment
redis # also a comment
Always the first item in the line, the source describes where the package can be found. A source can be any of the following things:
.rip
fileMore details on each under SOURCES
.
The version is any argument which starts with [a-z0-9]. Some examples:
See Fake Version
below to learn how to lie about the package
version.
A path is an argument which begins with a slash. Paths tells Rip which subdirectory to use as the base directory when installing the target package. For example, given this line:
git://github.com/rails/rails.git /activerecord
Rip would fetch and unpack git://github.com/rails/rails.git
but use
activerecord
as the root when installing.
A dependency can be specified using a star. It will be installed before the package which depends on it.
For example, these lines:
sinatra 0.9.4
* rack 1.0
Will basically be executed as:
$ rip-install rack 1.0
$ rip-install sinatra 0.9.4
Dependencies can be nested as deeply as you'd like.
sinatra 0.9.4
* rack 1.0
mustache 0.3.1
* temple 1.2
** hot-parser 0.4.5
** cacher 0.1
*** httparty 1.0 /party
* markdown 1.0
** rdiscount <IP_ADDRESS>
Rip derives a package's name from its source, but it may be helpful to specify your own name.
A name is an argument which begins with an equal sign followed by a greater than sign.
git://github.com/ezmobius/redis-rb.git =>redis
This line says we want the installed package to be named "redis". Note now that installing the gem "redis" will fail due to a name conflict.
This also means gems depending on the "redis" gem will see your git-based package as that depenedency, and may fail to install because the versions differ.
If that's the case you can specify a fake version.
Fake verisions register a package as being installed at a different version than it's actually installed at.
For example:
git://github.com/defunkt/mustache.git @1.0.2
This says mustache is installed at version 1.0.2, but really it'll
install the latest version from the git repo's master
branch.
This can be useful in development or when combined with the name
argument:
git://github.com/ezmobius/redis-rb.git @1.0 =>redis
This makes it appear as though we have version 1.0 of the redis gem installed.
Thanks to pip(1) for the inspiration.
Rip will use git to fetch a package if its source is in one of the following formats:
git://git.myproject.org/MyProject
git@git.myproject.org:MyProject.git
git+http://git.myproject.org/MyProject
<EMAIL_ADDRESS>Rip will also try to fetch any source ending in .git
:
http://git.myproject.org/MyProject.git
file:///Users/chris/Projects/rip/.git
Rip will use hg to fetch a package if its source is in one of the following formats:
hg+http://hg.myproject.org/MyProject/
hg+https://hg.myproject.org/MyProject/
<EMAIL_ADDRESS>Rip will use bzr to fetch a package if its source is in one of the following formats:
bzr+http://bzr.myproject.org/MyProject/trunk/ /MyProject
<EMAIL_ADDRESS>/MyProject
<EMAIL_ADDRESS>/MyProject
<EMAIL_ADDRESS>/MyProject
.rip
fileAny source ending in .rip
will be loaded recursively. That is, each
line will be fed to rip-install
as if they were a top level
line. Kind of like a partial or include.
deps.rip
http://defunkt.github.com/hub/latest.rip
Keep in mind these also work as argument to rip-install
:
$ rip-install http://defunkt.github.com/hub/latest.rip
Rip can run shell commands, it's true. Shell commands are special and take up the entire line - everything will be run right on the shell.
!brew install redis
!brew install mongo
!brew install ruby
redis 1.0
mongo
Anything ending in .zip
will be fetched and unzipped, be it remote
or local.
http://defunkt.github.com/backups/redis-rb.zip
Anything ending in .tar
, .tar.gz
, or `.tgz. will be fetched and
dezipped, be it remote or local.
http://defunkt.github.com/backups/redis.tar.gz
Directories can be specified as long as they begin with /
or ~
.
/var/packages/iphone
~/Projects/stunners @1.0
Files can be specified as long as they begin with /
or ~
.
/var/packages/iphone/iphone.rb
~/Projects/stunners/index.rb
http://github.com/defunkt/rip/issues
Chris Wanstrath<EMAIL_ADDRESS>rip(1), git(1), http://github.com, http://github.com/defunkt/rip


Select one:
First
This is second option
This is not second option
Fourth with spaces inside
Fifth surrounded by spaces
Select multiple:
First
This is second option
This is not second option
Fourth with spaces inside
Fifth surrounded by spaces
Select one from input:
Select multiple from input:


I am a backend developer with experience designing and building highly available, distributed systems that serve APIs. My experience encompasses designing, building and maintaining APIs that are available worldwide. Most of my professional experience is with Java, but I have a working knowledge of Python and Javascript.
My main personal project is focused around Android Development. I am currently working on porting an iOS app to Android. Additionally, I have started exploring different front end frameworks to add to my technical toolkit.
Currently working on creating Android version of Draftwise, a Fantasy Sports optimization App. The iOS version currently available on the App Store. Look for it on the Google Play store in the coming weeks! https://draftwise.io/


My First Website
This is the first website i am going to make. It will teach me the Basics ofhtmlandcss
HTML Learning Goals
- Page Structure
- Element Attributes
- Tag Types
- Linking in other resources
CSS Learning Goals
- What properties are
- How you use elements, and element attributes to change styles
- How CSS styles cascade
- How to include these Styles
| TH1 |
TH2 |
TH3 |
TH4 |
| TD11 |
TD12 |
TD13 |
TD14 |
| TD21 |
TD22 |
TD23 |
TD24 |
| TD31 |
TD32 |
TD33 |
TD34 |


Badge
Badge example below
After the new message prompt and summary information, unified on the right side of the list
Single-row list
details
Unread red dot follows the subject information and is unified on the left side of the list
8
Contact Name
System Information
Single-row list
8
Single-row list
8
details
Single-row list
New


Container Hierarchies
Unity container provides a way to create child containers (other systems refer to it as resolution scopes) and allows building sophisticated scope trees of registrations. There are just a few simple rules to follow when dealing with container hierarchies:
Types registered in predecessor containers are always available in descendant containers. This is a very simple concept, each registration is like a public virtual declaration in cs types. Every descendant inherits it and can use at will.
Types registered in descendant containers override the same registration of predecessors. Following the same analogy with public virtual declarations, each override registration installs its own declaration and hides the one in predecessor containers.
Using Container Hierarchies
Unity supports nested containers, allowing you to build container hierarchies. Nesting containers enables you to control the scope and lifetime of singleton objects, and register different mappings for specific types. This topic contains the following sections that describe how you can create container hierarchies and use them in your applications:
- Constructing and Disposing Unity Containers
- Controlling Object Scope and Lifetime
- Overriding registrations
Constructing and Disposing Unity Containers
The following methods enable you to create a new default UnityContainer, create a child container that has a specified UnityContainer as its parent, and dispose an existing container.
| Method | Description |
|---|---|
new UnityContainer() |
Creates a root UnityContainer. Returns a reference to the new container. |
CreateChildContainer() |
Creates a new nested UnityContainer as a child of the current container. The current container first applies its own settings, and then it checks the parent for additional settings. Returns a reference to the new container. |
Dispose() |
Disposes this container instance and any child containers. Also disposes any registered object instances whose lifetimes are managed by the container. |
Controlling Object Scope and Lifetime
When the container creates singleton objects, it manages the lifetime of these singletons. They remain in scope until you (or the garbage collector) dispose the container. At this point, it disposes the registered singleton instances it contains. In addition, if you dispose the parent container in a nested container hierarchy, it automatically disposes all child containers and the registered singletons they contain.
Therefore, if you require two separate sets of such objects that must have different lifetimes, you can use hierarchical containers to store and manage each set. Register instances that you want to be able to dispose separately in one or more child containers that you can dispose without disposing the parent container.
Note that due to possibility of child container disposal, resolution of singletons (registered with Singleton
or PerContainer
lifetime) will only be using registrations available in the container they were registered in and its ancestors, but not child containers, to satisfy their dependencies, even if the resolution is requested through a child container. It's Unity's way of making sure it is not storing an instance that references potentially disposed objects.
The following code demonstrates the use of a child container to manage the lifetime of specific singleton instances while maintaining the singleton instances in the parent container.
// Create parent container
IUnityContainer parentCtr = new UnityContainer();
// Register type in parent container
parentCtr.RegisterType<MyParentObject>(TypeLifetime.PerContainer);
// Create nested child container in parent container
IUnityContainer childCtr = parentCtr.CreateChildContainer();
// Register type in child container
childCtr.RegisterType<MyChildObject>(TypeLifetime.PerContainer);
// Create instance of type stored in parent container
MyParentObject parentObj = parentCtr.Resolve<MyParentObject>();
// Create instance of type stored in child container
MyChildObject childObj = childCtr.Resolve<MyChildObject>();
// ... can use both generated objects here ...
// Dispose child container
childCtr.Dispose();
// ... can use only object in parent container here ...
// Dispose parent container
parentCtr.Dispose();
Overriding registrations
You can use nested containers when you have slightly different dependency injection requirements for specific objects but want to provide a fallback facility for objects that implement a specific interface or are of a specific type. For example, you may have a general requirement for objects that implement the IMyObject interface to map to the type MyStandardObject. However, in specific parts of the application code, you may want the IMyObject interface to map to the type MySpecialObject.
In this case, you can register the general mapping in the parent container and register the specific case in a child container. Then, when you want to obtain an instance of the object, you call the Resolve method on the appropriate container. If you call the method on the child container, it returns an object of type MySpecialObject. If you call the method on the parent container, it returns an object of type MyStandardObject.
However, the advantage with nested containers is that, if the child container cannot locate a mapping for the requested interface or type, it passes the request to its parent container and onward through the hierarchy until it reaches the root or base container. Therefore, for objects not mapped in the child container, the mapping in the parent container (or in an ancestor container where there are more than two levels in the hierarchy) defines the object type returned.
The following code shows how you can implement the preceding scenario.
// Create parent container
IUnityContainer parentCtr = new UnityContainer();
// Register two mappings for types in parent container
parentCtr.RegisterType<IMyObject, MyStandardObject>();
parentCtr.RegisterType<IMyOtherObject, MyOtherObject>();
// Create nested child container in parent container
IUnityContainer childCtr = parentCtr.CreateChildContainer();
// Register mapping for specific type in child container
childCtr.RegisterType<IMyObject, MySpecialObject>();
// Now retrieve instances of the mapped objects using the child container.
// Using the interface as the type for the returned objects means that it
// does not matter which container returns the actual object.
// This code returns an object of type MySpecialObject using the mapping
// registered in the child container:
IMyObject specialObject = childCtr.Resolve<IMyObject>();
// This code returns an object of type MyOtherObject using the mapping
// registered in the parent container because there is no mapping in
// the child container for this type:
IMyOtherObject otherObject = childCtr.Resolve<IMyOtherObject>();
// Now retrieve instance of the standard object using the parent container.
// This code returns an object of type MyStandardObject using the mapping
// registered in the parent container:
IMyObject standardObject = parentCtr.Resolve<IMyObject>();
// Dispose parent container and child container
parentCtr.Dispose();


CNA Class
Theory studies and clinical preparation for State nursing assistant certifcation.
This last and highly used version of my online CNA training, which existed from December 2006 thru May 2017,
was the first private, independently-operated CNA program in Montana. It brought me goal-oriented students from across MT to finish the course with
their "hand-on" clinical skills & MT certification exam in my lab and in my assisted living facility. The class utilized other sites, including:
Youtube, Google Docs, Scribd, StudyStack and KeepAndShare. The site ranked high on the first page in google searches while in its best years,
and all that was required for the class was accessible from this page.
- Date: August 2013-May 2017
- Client: Heart and Hands CNA Training
- Category: Education Site


{% extends "base.html" %} {% block title %}Current status{% endblock %} {% block content %}
Please select a region to see how clean its electricity is right now. (Currently supported states are in green.)


Lorem ipsum dolor sit amet, consectetur adipisicing elit. Quisquam dolore sequi officia minima delectus nesciunt tempora libero culpa eum id, nostrum possimus, corporis atque veritatis doloremque numquam omnis facere consequuntur?
Building your project
Build your project easily with Gulp. There are tasks available for building html, scss, javascript and images. Code quality will be checked by jshint. All you have to do is:
gulp build
Work on your project locally and watch your changes using BrowserSync:
gulp watch
Deploy your project to Github Pages. By using the follow command, your project dist folder will be send to the gh-pages branch.


Gloria Rated (R) By James Sanford The Kalamazoo Gazette
Roger Ebert has often brought up the idea that instead of rehashing the classics, filmmakers should concentrate on revising movies that weren't very good to begin with. Hollywood may have taken his comments to heart.
Director Terrence Malick's "The Thin Red Line" is a loose remake of the unsuccessful 1964 film of the same name, and now director Sidney Lumet has tackled "Gloria," based on a 1980 melodrama that was nothing to shout about, but did feature a memorably flinty, Oscar-nominated performance by Gena Rowlands.
And what are the results of these experiments? Malick's epic is flawed but intermittently fascinating, rife with luscious imagery. "Gloria," on the other hand, finds Sharon Stone doing her darnedest to squeeze into Rowlands' high heels, and it doesn't take long to see they don't become her.
Rowlands' Gloria was a bitter ex-gangster's moll whose heart had long ago gone into a deep-freeze. Stone's Gloria, aside from the occasional flip remark or crude comment, turns out to be a soft-shell crab. For a woman who's supposedly so street-seasoned, she's weirdly skittish and sentimental.
The reason for her transformation from bad mutha to mother figure is Nicky Nunez (Jean-Luke Figueroa), a 7-year-old whose family was been massacred by the henchmen of Gloria's ex-boyfriend Kevin (Jeremy Northam), minutes after Nicky's dad sent the kid off with a valuable computer disk and a teary "you da man." For reasons never made clear, the supposedly steely Gloria steals Nicky from Kevin's clutches and gives him a tour of New York, all the while dodging the bad guys who want that precious disk.
Unable to commit to being a thriller or a character study, the movie settles for being an excitement-free drag. Lumet, whose talent peaked 25 years ago with "Dog Day Afternoon" and "Network," throws in one amazingly sloppy car chase, but otherwise does nothing to perk up the picture's sluggish pace.
Stone's work here has two modes, shrill and mushy, and her Noo Yawk accent quickly unravels; in one scene she's talking about having been in jail for "twee" years, while in the next she sharply pronounces the "h" in "therapy." Bad dialects also hobble Northam and even George C. Scott, who turns up briefly as one of Gloria's former sugar daddies.
If there's anything to be said in the movie's favor, it's that "Gloria" offers a few glimmers of the natural wit Stone demonstrated during her recent appearance on Bravo's "Inside the Actors' Studio." After Nicky, who's shared her motel bed the night before, tells Gloria he likes sleeping with her, Stone delivers her reply - "You're not the first guy to tell me that" - with a smug sneer that would have made Mae West proud. James Sanford
The review above was posted to the
rec.arts.movies.reviews newsgroup (de.rec.film.kritiken for German reviews).
The Internet Movie Database accepts no responsibility for the contents of the
review and has no editorial control. Unless stated otherwise, the copyright
belongs to the author.
Please direct comments/criticisms of the review to relevant newsgroups.
Broken URLs inthe reviews are the responsibility of the author.
The formatting of the review is likely to differ from the original due
to ASCII to HTML conversion.
Related links: index of all rec.arts.movies.reviews reviews


| Created: | 6/27/2020 7:02:37 AM |
| Modified: | 6/27/2020 7:02:37 AM |
| Project: | |
| Advanced: |
| Attribute |
|
Public serial id |
|
Details:
|
|
Public varchar(255) header1 |
|
Details:
|
|
Public varchar(255) header2 |
|
Details:
|
|
Public varchar(255) header3 |
|
Details:
|
|
Public varchar(255) header4 |
|
Details:
|
|
Public integer rank |
|
Details:
|
|
Public integer fdr_appropriation_reference_id |
|
Details:
|
|
Public boolean is_federal |
|
Details:
|
|
Public varchar(255) key |
|
Details:
|
|
Public timestamp with time zone(0) start_date |
|
Details:
|
|
Public timestamp with time zone(0) end_date |
|
Details:
|
|
Public varchar(255) created_by |
|
Details:
|
|
Public varchar(255) updated_by |
|
Details:
|
|
Public timestamp with time zone(0) created_at |
|
Details:
|
|
Public timestamp with time zone(0) updated_at |
|
Details:
|
| Operation | ||
|
Public buckets_pkey( id: serial, |
||
|
Details:
<<PK>>
|
| Element | Source Role | Target Role |
|
«table» bucket_versions Table «FK» |
Name: bucket_versions_bucket_id_foreign |
Name: buckets_pkey |
|
Details:
|
| Tag | Value |
| Owner | public |
|
Details:
|
| Property | Value |
| isFinalSpecialization: | 0 |


The following ads and images contain an image sourced from wikimedia commons (https://commons.wikimedia.org/wiki/File:Parliament_House_Canberra_NS.jpg) under CC-BY-SA 3.0. They are redistributed under the same license.


EYE is a reasoning engine supporting the Semantic Web layers.
It performs controlled chaining and it supports Euler paths.
Via N3 it is interoperable with Cwm.
Controlled chaining is backward chaining for rules using <=
in N3
and forward chaining for rules using =>
in N3.
This can be seen at work in EYE reasoning.
Euler paths are roughly "don't step in your own steps" which is inspired by
what Leonhard Euler discovered in 1736 for the Königsberg Bridge Problem.
EYE sees the rule P => C
as P & NOT(C) => C
.
EYE can be installed manually on Linux, Windows and MacOSX.
EYE is also available in a Docker container for command line use
and in a Docker container for HTTP client use.
The EYE stack comprises the following Software and Machines:
This is what the basic EAM (Euler Abstract Machine) does in a nutshell: