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To date, the US Department of Energy's Joint Genome Institute has produced shotgun libraries for more than 150 microbial whole genome sequencing projects. In an effort to better span repeats and link contigs we now utilize a 3 library approach for all whole genome shotgun projects. Here, we describe the construction, sequencing, and analysis of our 3 library cloning approach with the goal of producing high quality draft assemblies with greater genome coverage and fewer gaps. The libraries were constructed from randomly sheared whole genomic DNA that was size selected to approximately 3 Kb, 8 Kb or 40 Kb and ligated into the appropriate high, low, or single copy cloning vector. The libraries were sequenced to an approximate depth of 4X for the 3 Kb and 8 Kb libraries, and approximately 15X clone coverage for the 40 Kb fosmid library. Analysis to date of a wide array of microbial projects indicates our cloning strategy has been successful in spanning repeat regions and producing longer-range contiguity. These assemblies reduce the amount of finishing required to complete the genome sequence.

Resistance represents a major challenge for antibody-based therapy for COVID-19.sup.1-4. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern--B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)--and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19. An engineered IgM antibody administered intranasally in mice shows high prophylactic efficacy and therapeutic efficacy against SARS-CoV-2, and is also effective against multiple variants of concern that are resistant to IgG-based therapeutics., Author(s): Zhiqiang Ku [sup.1] , Xuping Xie [sup.2] , Paul R. Hinton [sup.3] , Xinli Liu [sup.4] , Xiaohua Ye [sup.1] , Antonio E. Muruato [sup.5] [sup.6] , Dean C. [...]

The US DOE Joint Genome Institute (JGI) is a high-throughput genomics facility involved in sequencing a variety of organisms. A major effort at JGI is the sequencing of genomes and microbial community samples of relevance to the DOE missions of carbon sequestration, bioremediation and energy production. cDNA/EST sequencing is an integral part of genomic sequencing because it provides crucial information for gene models and genome annotation. The 454 sequencing platform is an integrated system of emulsion-based PCR amplification of hundreds of thousands of DNA fragments linked to high throughput parallel pyrosequencing in picoliter-sized wells. Several strategies have been designed and carried out at JGI to use the 454 platform for cDNA/EST sequencing. cDNA libraries constructed by conventional methods were subjected to direct 454 sequencing. In addition, special primers and adaptors were also designed for library construction so the directional sequencing feature of the 454 technology can be used to sequence a particular end of the cDNA/EST fragments. Adaptor sequences used by 454 library construction can be incorporated into polyT primer, cap primer and/or random primer for cDNA/EST library construction. The 454 sequencing platform can deliver 200 to 400 thousand cDNA/EST reads from a single run and does not require cloning step, potentially improving the coverage obtained through traditional Sanger sequencing. The large numbers of short reads generated by the 454 platform can be aligned to genome assemblies to extend and confirm gene models. Results from different strategies of library construction combined with 454 sequencing will be presented. The coverage of the library and the novelty rate are compared with traditional Sanger sequencing. The possible assembly problems caused by short reads with slightly higher error rate from 454 will also be addressed.

Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of skin. Its expression is stimulated by a number of agents including calcium and 1,25-dihydroxy-vitamin D3 that promote the differentiation process in keratinocytes. Within the distal regulatory region of the involucrin promoter lies an AP-1 site and an element homologous to other vitamin D response elements. In previous studies mutation of the AP-1 site was found to reduce basal activity and block calcium stimulation of the involucrin promoter, whereas the vitamin D response element was not critical for calcium regulation. In this study both elements proved to be important for 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. Mutation of the AP-1 site reduced basal activity and blocked 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. In contrast, mutation of the vitamin D response element did not reduce basal expression of the involucrin promoter or prevent calcium stimulation of involucrin gene expression, but blocked 1,25-dihydroxyvitamin D3 stimulation. The vitamin D response element from the involucrin gene bound the vitamin D receptor and the retinoid X receptor, but not the retinoic acid receptor, in a specific manner. We conclude that the AP-1 site and the vitamin D response element in the involucrin promoter play important roles in mediating the action of 1,25-dihydroxyvitamin D3 on involucrin expression, but the vitamin D response element provides specificity for the 1,25-dihydroxyvitamin D3 response lacking at the AP-1 site. [ABSTRACT FROM AUTHOR]

Calcium induces both involucrin and transglutaminase-K in normal keratinocytes (NHK) but not in squamous carcinoma cell lines (SCC). The protein kinase C (PKC) agonist phorbol myristoyl acetate potentiates and the PKC antagonist Ro31-8220 blocks the ability of calcium to stimulate the involucrin promoter in normal human keratinocytes but not in SCC4. We thus examined the ability of calcium to regulate the levels of five PKC isozymes in NHK and two SCC. In the normal keratinocytes, the levels of PKC [alpha], PKC [delta], PKC [eta], and PKC [zeta] increased over the first one to two weeks in a calcium-and time-dependent manner. PKC [epsilon] decreased in a time-and calcium-dependent fashion over the three-week period. All five isozymes showed little change during culture in SCC4 at any calcium concentration. Calcium and time of culture had partial effects on SCC12B2, a carcinoma that shows partial differentiation characteristics. Since PKC [alpha] is the only calcium responsive PKC isozyme in keratinocytes and most likely to be directly involved in calcium induced differentiation, we evaluated the effect of inhibiting its production with antisense oligonucleotides on calcium-regulated markers of differentiation. We found that the PKC [alpha] specific antisense oligonucleotide blocked calcium stimulated involucrin promoter activity as well as PKC [alpha], involucrin, and transglutaminase protein production, whereas the sense oligonucleotide control did not. We conclude that although a number of PKC isozymes are regulated during calcium-induced differentiation, PKC [alpha] plays a necessary role in mediating calcium-induced differentiation. Failure to regulate PKC [alpha] in SCC4 may underlie at least part of the failure of calcium to promote differentiation in these cells., (Copyright 2003 Wiley-Liss, Inc.)