Identification of antibody-resistant SARS-CoV-2 mutants via N4-Hydroxycytidine mutagenesis.

Monoclonal antibodies targeting the Spike protein of SARS-CoV-2 are effective against COVID-19 and might mitigate future pandemics. However, their efficacy is challenged by the emergence of antibody-resistant virus variants. We developed a method to efficiently identify such resistant mutants based on selection from mutagenized virus pools. By inducing mutations with the active compound of Molnupiravir, N4-hydroxycytidine (NHC), and subsequently passaging the virus in the presence of antibodies, we identified specific Spike mutations linked to resistance. Validation of these mutations was conducted using pseudotypes and immunofluorescence analysis. From a Wuhan-like strain of SARS-CoV-2, we identified the following mutations conferring strong resistance towards the corresponding antibodies: Bamlanivimab - E484K, F490S and S494P; Sotrovimab - E340K; Cilgavimab - K444R/E and N450D. From the Omicron B.1.1.529 variant, the strongly selected mutations were: Bebtelovimab - V445A; Sotrovimab - E340K and K356M; Cilgavimab - K444R, V445A and N450D. We also identified escape mutations in the Wuhan-like Spike for the broadly neutralizing antibodies S2K146 - combined G485S and Q493R - and S2H97 - D428G, K462E and S514F. Structural analysis revealed that the selected mutations occurred at antibody-binding residues within the receptor-binding domains of the Spike protein. Most of the selected mutants largely maintained ACE2 binding and infectivity. Notably, many of the identified resistance-conferring mutations are prevalent in real-world SARS-CoV-2 variants, but some of them (G485S, D428G, and K462E) have not yet been observed in circulating strains. Our approach offers a strategy for predicting the therapeutic efficacy of antibodies against emerging virus variants.

Unveiling the Brazilian kefir microbiome: discovery of a novel Lactobacillus kefiranofaciens (LkefirU) genome and in silico prospection of bioactive peptides with potential anti-Alzheimer properties.

BACKGROUND: Kefir is a complex microbial community that plays a critical role in the fermentation and production of bioactive peptides, and has health-improving properties. The composition of kefir can vary by geographic localization and weather, and this paper focuses on a Brazilian sample and continues previous work that has successful anti-Alzheimer properties. In this study, we employed shotgun metagenomics and peptidomics approaches to characterize Brazilian kefir further. RESULTS: We successfully assembled the novel genome of Lactobacillus kefiranofaciens (LkefirU) and conducted a comprehensive pangenome analysis to compare it with other strains. Furthermore, we performed a peptidome analysis, revealing the presence of bioactive peptides encrypted by L. kefiranofaciens in the Brazilian kefir sample, and utilized in silico prospecting and molecular docking techniques to identify potential anti-Alzheimer peptides, targeting ß-amyloid (fibril and plaque), BACE, and acetylcholinesterase. Through this analysis, we identified two peptides that show promise as compounds with anti-Alzheimer properties. CONCLUSIONS: These findings not only provide insights into the genome of L. kefiranofaciens but also serve as a promising prototype for the development of novel anti-Alzheimer compounds derived from Brazilian kefir.

High-resolution HLA genotyping improves PIRCHE-II assessment of molecular mismatching in kidney transplantation.

HLA matching in solid organ transplant is performed with the aim of assessing immunologic compatibility in order to avoid hyperacute rejection and assess the risk of future rejection events. Molecular mismatch algorithms are intended to improve granularity in histocompatibility assessment and risk stratification. PIRCHE-II uses HLA genotyping to predict indirectly presented mismatched donor HLA peptides, though most clinical validation studies rely on imputing high resolution (HR) genotypes from low resolution (LR) typing data. We hypothesized that use of bona fide HR typing could overcome limitations in imputation, improving accuracy and predictive ability for donor-specific antibody development and acute rejection. We performed a retrospective analysis of adult and pediatric kidney transplant donor/recipient pairs (N = 419) with HR typing and compared the use of imputed LR genotyping verses HR genotyping for PIRCHE-II analysis and outcomes. Imputation success was highly dependent on the reference population used, as using historic Caucasian reference populations resulted in 10 % of pairs with unsuccessful imputation while multiethnic reference populations improved successful imputation with only 1 % unable to be imputed. Comparing PIRCHE-II analysis with HR and LR genotyping produced notably different results, with 20 % of patients discrepantly classified to immunologic risk groups. These data emphasize the importance of using multiethnic reference panels when performing imputation and indicate HR HLA genotyping has clinically meaningful benefit for PIRCHE-II analysis compared to imputed LR typing.

Next-Generation DNA Sequencing of Grade 1 Meningioma Tumours: A Case Report of Angiomatous and Psammomatous Meningiomas.

We performed the next-generation sequencing (NGS) analysis of a rare grade 1 brain meningioma (angiomatous type) and a common grade 1 spinal meningioma (psammomatous type) and compared their mutation profiling. The data were analysed using the Ion Reporter 5.16 programme (Thermo Fisher Scientific, Waltham, MA). Sequencing analysis identified 10 novel variants and two previously reported variants that were common between these two tumours. Nine variants were missense, which included an insertion in EGFR c.1819_1820insCA, causing frameshifting, and a single nucleotide deletion in HRAS and HNF1A genes, causing frameshifting in these genes. These were common variants identified for both tumours. Also, 10 synonymous variants and 10 intronic variants were common between these two tumours. In intronic variants, two were splice site_5' variants (acceptor site variants). Typical of the angiomatous type tumour, there were 11 novel and six previously reported variants that were not found in the psammomatous tumour; three variants were synonymous, 11 were missense mutations, and three were deletions causing frameshifting. The deletion variants were in the SMARCB1, CDH1, and KDR genes. In contrast, eight novel and five previously reported variants were found in the psammomatous meningioma tumour. In this tumour, two variants were synonymous: a deletion causing a frameshifting in [(c.3920delT; p. (Ile1307fs)], and a two-base pair insertion and deletion (INDEL) [(c.3986_3987delACinsGT; p. (His1329Arg)] both in the APC gene were also found. Among our findings, we have identified that ALK, VHL, CTNNB1, EGFR, ERBB4, PDGFRA, KDR, SMO, ABL1, HRAS, ATM, HNF1A, FLT3, and RB1 mutations are common for psammomatous meningioma and angiomatous tumours. Variants typical for angiomatous (brain) meningioma are PIK3CA, KIT, PTPN11, CDH1, SMAD4, and SMARCB1; the variants typical for psammomatous meningioma are APC, FGFR2, HNF1A, STK11, and JAK3. The RET splice variant (c.1880-2A>C) found in both meningioma tumours is reported (rs193922699) as likely pathogenic in the Single Nucleotide Polymorphism Database (dbSNP). All missense variants detected in these two meningiomas are found in the cancer-driver genes. The eight variants we found in genes such as EGFR, PDGFRA, SMO, FLT3, PIK3CA, PTPN11, CDH1, and RB1 are glioma-driver genes. We did not find any mutations in genes such as BRAF, IDH1, CDKN2A, PTEN, and TP53, which are also listed as cancer-driver genes in gliomas. Mutation profiling utilising NGS technology in meningiomas could help in the accurate diagnosis and classification of these tumours and also in developing more effective treatments.

Endomicrobiome of in vitro and natural plants deciphering the endophytes-associated secondary metabolite biosynthesis in Picrorhiza kurrooa, a Himalayan medicinal herb.

IMPORTANCE: Picrorhiza kurrooa is a major source of picrosides, potent hepatoprotective molecules. Due to the ever-increasing demands, overexploitation has caused an extensive decline in its population in the wild and placed it in the endangered plants' category. At present plant in-vitro systems are widely used for the sustainable generation of P. kurrooa plants, and also for the conservation of other commercially important, rare, endangered, and threatened plant species. Furthermore, the in-vitro-generated plants had reduced content of therapeutic secondary metabolites compared to their wild counterparts, and the reason behind, not well-explored. Here, we revealed the loss of plant-associated endophytic communities during in-vitro propagation of P. kurrooa plants which also correlated to in-planta secondary metabolite biosynthesis. Therefore, this study emphasized to consider the essential role of plant-associated endophytic communities in in-vitro practices which may be the possible reason for reduced secondary metabolites in in-vitro plants.

Applications of High-Throughput DNA Sequencing to Single-Domain Antibody Discovery and Engineering.

Next-generation DNA sequencing (NGS) technologies have made it possible to interrogate antibody repertoires to unprecedented depths, typically via sequencing of cDNAs encoding immunoglobulin variable domains. In the absence of heavy-light chain pairing, the variable domains of heavy chain-only antibodies (HCAbs), referred to as single-domain antibodies (sdAbs), are uniquely amenable to NGS analyses. In this chapter, we provide simple and rapid protocols for producing and sequencing multiplexed immunoglobulin variable domain (VHH, VH, or VL) amplicons derived from a variety of sources using the Illumina MiSeq platform. Generation of such amplicon libraries is relatively inexpensive, requiring no specialized equipment and only a limited set of PCR primers. We also present several applications of NGS to sdAb discovery and engineering, including: (1) evaluation of phage-displayed sdAb library sequence diversity and monitoring of panning experiments; (2) identification of sdAbs of predetermined epitope specificity following competitive elution of phage-displayed sdAb libraries; (3) direct selection of B cells expressing antigen-specific, membrane-bound HCAb using antigen-coupled magnetic beads and identification of antigen-specific sdAbs, and (4) affinity maturation of lead sdAbs using tandem phage display selection and NGS. These methods can easily be adapted to other types of proteins and libraries and expand the utility of in vitro display technology.

Variability of longitudinal triglyceride phenotype in patients heterozygous for pathogenic APOA5 variants.

BACKGROUND: Biallelic pathogenic variants in APOA5 are an infrequent cause of familial chylomicronemia syndrome characterized by severe, refractory hypertriglyceridemia (HTG), and fasting plasma triglyceride (TG) >10 mmol/L (>875 mg/dL). The TG phenotype of heterozygous individuals with one copy of a pathogenic APOA5 variant is less familiar. We evaluated the longitudinal TG phenotype of individuals with a single pathogenic APOA5 variant allele. METHODS: Medically stable outpatients from Ontario, Canada were selected for study based on having: 1) a rare pathogenic APOA5 variant in a single allele; and 2) at least three serial fasting TG measurements obtained over >1.5 years of follow-up. RESULTS: Seven patients were followed for a mean of 5.3 ± 3.7 years. Fasting TG levels varied widely both within and between patients. Three patients displayed at least one normal TG measurement (<2.0 mmol/L or <175 mg/dL). All patients displayed mild-to-moderate HTG (2 to 9.9 mmol/L or 175 to 875 mg/dL) at multiple time points. Five patients displayed at least one severe HTG measurement. 10%, 54%, and 36% of all TG measurements were in normal, mild-to-moderate, and severe HTG ranges, respectively. CONCLUSIONS: Heterozygosity for pathogenic variants in APOA5 is associated with highly variable TG phenotypes both within and between patients. Heterozygosity confers susceptibility to elevated TG levels, with secondary factors likely modulating the phenotypic severity.

Supratentorial Sporadic Hemangioblastoma: A Case Report With Mutation Profiling Using Next-Generation DNA Sequencing.

The present study aimed to determine genomic changes in sporadic intracranial hemangioblastoma (HBL), and the mutation patterns were analyzed using next-generation DNA sequencing (NGS). In this NGS analysis of the HBL tumor, 67 variants of 41 genes were identified. Of these, 64 were single-nucleotide variants (SNVs), two were exonic insertions and deletions (INDEL), and one was an intronic INDEL. In total, 15 were missense exonic variants, including an insertion variant in the NRAS gene, c.1_2insA, and a deletion variant, c.745delT, in the HNF1A gene, both of these mutations produced a termination codon. Other exonic missense variants found in the tumor were CTNNB1, FGFR3, KDR, SMO, HRAS, RAI1, and a TP53 variant (c.430C>G). Moreover, the results of the present study revealed a novel variant, c.430C>G, in TP53 and two missense variants of SND1 (c.1810G>C and c.1814G>C), which were also novel. ALK (rs760315884) and FGFR2 (rs1042522) missense variants were reported previously. Notably, a total of 10 previously reported single-nucleotide polymorphisms (SNPs) were found in this tumor in genes including MLH1 (rs769364808), FGFR3 (rs769364808), two variants (rs1873778 and rs2228230) in PDGFRA, KIT (rs55986963), APC (rs41115), and RET (rs1800861). The results of this study revealed a synonymous mutation (SNP) in c.1104 G>T; p. (Ser368Ser) in the MLH1 gene. In this amino acid (AA) codon, two other variants are also known to cause missense substitutions, c.1103C>G; p. (Ser368Trp); COSM6986674) and c.1103C>T; p.(Ser368Leu; COSM3915870), were found in hematopoietic and urinary tract tissue, respectively. However, three SNPs found in genes such as ALK, KDR, and ABL1 in the HBL tumor in this study were not reported in UCSC, COSMIC, and ClinVar databases. Additionally, 19 intronic variants were identified in this tumor. One intronic SNV was present in each of the following genes: EGFR, ERBB4, KDR, SMO, CDKN2B, PTEN, PTPN11, RB1, AKT1, and ERBB2. In PIK3CA and FBXL18 genes, two intronic variants were present, and in the SND1 gene, three intronic variants were detected in the HBL tumor presented in this study. Notably, only one of these was reported in the catalog of somatic mutations in cancer. Only one 3'-untranslated region (UTR) insertion variant in the NRAS gene (c.*2010T>AT) was detected in the tumor of the present study, and this was a splice site acceptor. A TP53 intronic mutation (c.782+1G>T) was the only pathogenic splice_donor_variant found in this HBL tumor. The frequency of variants and Phred scores were markedly high, and the p-values were significant for all of the aforementioned mutations. In summary, a total of 15 missense, 10 synonymous, and 19 intronic variants were identified in the HBL tumor. Results of the present study detected one novel insertion in NRAS and one novel deletion in HNF1A genes, a novel missense variant in the TP53 gene, and two novel missense variants of SND1. Hotspot mutations in other cancer driver genes, such as PTEN, ATM, SMAD4, SMARCB1, STK11, NPM1, CDKN2A, and EGFR, which are frequently affected in gliomas, were not found in the tumor of the present study. Future studies should aim to validate oncogenic mutations that may act as novel targets for the treatment of these tumors.

Contemporary advances in the endoscopic management of cholangiocarcinoma: a review of accomplished milestones and prospective opportunities.

INTRODUCTION: Cholangiocarcinoma, a primary malignancy of epithelial cells of the bile ducts, has been shown to have increasing incidence rates globally. Many of the current advances aim to improve the accuracy of differentiation between benign biliary strictures and cholangiocarcinoma, which include endoscopic techniques, devices, image processing, and the use of genomic sequencing in acquired specimens. AREAS COVERED: In this review, the authors explore the historical timeline of changes leading to modern management of cholangiocarcinoma, with special emphasis on endoscopic modalities and novel therapeutic interventions. The authors also expand on the strengths and shortcomings of endoscopic diagnostics and techniques in biliary drainage and finally discuss potential areas to focus for future research and development. EXPERT OPINION: Despite the advances in diagnosis and management of cholangiocarcinoma, there remain multiple tasks that are still awaiting to be completed. Next-generation sequencing in the diagnosis of cholangiocarcinoma needs to be further tested, validated, and easily obtainable. Other innovative diagnostic modalities, such as the use of artificial intelligence in cholangioscopy, may provide an effective complementary modality to existing techniques. A consensus on biliary drainage needs to be defined and account for longevity and patient convenience.

Technical feasibility of newborn screening for spinal muscular atrophy by next-generation DNA sequencing.

Newborn screening (NBS) assays for spinal muscular atrophy (SMA) typically use a polymerase chain reaction (PCR) based assay to identify individuals with homozygous deletion in exon 7 of the SMN1 gene. Due to high DNA sequence homology between SMN1 and SMN2, it has previously been difficult to accurately bioinformatically map short reads from next-generation DNA sequencing (NGS) to SMN1, resulting in low analytical performance and preventing NGS being used for SMA screening. Advances in bioinformatics have allowed NGS to be used in diagnostic settings, but to date these assays have not reached the scale required for high volume population newborn screening and have not been performed on the dried blood spot samples that NBS programs currently use. Here we integrate an NGS assay using hybridisation-based capture with a customised bioinformatics algorithm and purpose designed high throughput reporting software into an existing NBS program to achieve a laboratory workflow for population SMA screening. We tested the NGS assay on over 2500 newborns born over 2 weeks in a NBS program in a technical feasibility study and show high sensitivity and specificity. Our results suggest NGS may be an alternate method for SMA screening by NBS programs, providing a multiplex testing platform on which potentially hundreds of inherited conditions could be simultaneously tested.

The longitudinal triglyceride phenotype in heterozygotes with LPL pathogenic variants.

BACKGROUND: Biallelic pathogenic variants in lipoprotein lipase (LPL) cause familial chylomicronemia syndrome with severe hypertriglyceridemia (HTG), defined as plasma triglycerides (TG) > 10 mmol/L (> 885 mg/dL). TG levels in individuals with one copy of a pathogenic LPL gene variant is less familiar; some assume that the phenotype is intermediate between homozygotes and controls. OBJECTIVE: We undertook an evaluation of the longitudinal TG phenotype of individuals heterozygous for pathogenic LPL variants. METHODS: Medically stable outpatients were evaluated based on having: (1) a single copy of a rare pathogenic LPL variant; and (2) serial fasting TG measurements obtained over > 1.5 years of follow-up. RESULTS: Fifteen patients with a single pathogenic LPL variant were followed for a mean of 10.3 years (range 1.5 to 30.3 years). TG levels varied widely both within and between patients. One patient had normal TG levels < 2.0 mmol/L (< 175 mg/dL) continuously, while four patients had at least one normal TG level. Most patients fluctuated between mild-to-moderate and severe HTG: five patients had only mild-to-moderate HTG, with TG levels ranging from 2.0 to 9.9 mmol/L (175 to 885 mg/dL), while 6 patients had at least one instance of severe HTG. Of the 203 total TG measurements from these patients, 14.8%, 67.0% and 18.2% were in the normal, mild-to-moderate and severe HTG ranges, respectively. CONCLUSION: The heterozygous LPL deficient phenotype is highly variable both within and between patients. Heterozygosity confers susceptibility to a wide range of TG phenotypes, with severity likely depending on secondary factors.

Proteomics and AQP2 regulation.

The advent of modern quantitative protein mass spectrometry techniques around the turn of the 21st century has contributed to a revolution in biology referred to as 'systems biology'. These methods allow identification and quantification of thousands of proteins in a biological specimen, as well as detection and quantification of post-translational protein modifications including phosphorylation. Here, we discuss these methodologies and show how they can be applied to understand the effects of the peptide hormone vasopressin to regulate the molecular water channel aquaporin-2. The emerging picture provides a detailed framework for understanding the molecular mechanisms involved in water balance disorders.

Impact of an Enhanced Infection Control Protocol on the Microbial Community Profile in Molar Root Canal Treatment: An in Vivo NGS Molecular Study.

INTRODUCTION: Recent findings demonstrated that 1-year cone-beam computed tomography-based outcomes of molar root canal treatment were improved through an enhanced infection protocol (EnP), when compared with a current best-practice standard infection control protocol (StP). The EnP comprised measures to reduce iatrogenic contamination from direct and indirect contact surfaces, including the replacement of the rubber dams, gloves, files, all instruments, and surface barriers before root canal obturation. The aim of this study was to investigate the effect of such an EnP on resident microbiome present after chemomechanical instrumentation and the protocol ability in reducing iatrogenic contamination in molar teeth during root canal treatment. METHODS: Molar teeth were block-randomized to receive treatment under EnP or StP. To compare the differential effect of the protocol on the identity of bacteria present, 150 matched DNA extracts from 75 molar teeth samples (StP, n = 39; EnP, n = 36) were evaluated. Samples were taken before (S1) and after (S2) chemomechanical preparation and were subjected to next-generation sequencing of the V3-V4 region of the 16S rRNA gene before bioinformatical identification using the HOMD oral microbiome database and downstream taxonomic processing, providing measures of richness and diversity of bacteria and significant bacterial taxa during chemomechanical instrumentation and the effect of the 2 treatment groups. RESULTS: Eighty-eight microbial taxa were significantly more abundant in StP S2 samples, including endodontically relevant contaminants taxa as Actinomyces, Cutibacterium, and Haemophilus. The S2 samples demonstrated fewer residual bacterial species in the EnP group, with 26.8 observed species compared with 38.3 in the StP. Reduced diversity and richness measures were noted in the EnP pre-obturation samples compared with the StP in OTU, Chao1, and ACE indices (P ≤ .05). Differential microbial identities between S1 and S2 samples and protocols demonstrated that the previously observed increased effectiveness of the EnP protocol was likely to prevent recontamination or de novo contamination of the root canal space during treatment. CONCLUSIONS: The implemented EnP resulted in a specific reduction of microbial taxa often associated with recontamination or iatrogenic contamination, suggesting the basis for improved infection control measures during root canal treatment.

MuSE: A Novel Approach to Mutation Calling with Sample-Specific Error Modeling.

Accurate detection of somatic mutations in genetically heterogeneous tumor cell populations using next-generation sequencing remains challenging. We have developed MuSE, Mutation calling using a Markov Substitution model for Evolution, a novel approach for modeling the evolution of the allelic composition of tumor and normal tissue at each reference base. It adopts a sample-specific error model to depict inter-tumor heterogeneity, which greatly improves the overall accuracy. Here, we describe the method and provide a tutorial on the installation and application of MuSE.

Molecular Diagnostic Tools Applied for Assessing Microbial Water Quality.

Microbial water quality is of vital importance for human, animal, and environmental health. Notably, pathogenically contaminated water can result in serious health problems, such as waterborne outbreaks, which have caused huge economic and social losses. In this context, the prompt detection of microbial contamination becomes essential to enable early warning and timely reaction with proper interventions. Recently, molecular diagnostics have been increasingly employed for the rapid and robust assessment of microbial water quality implicated by various microbial pollutants, e.g., waterborne pathogens and antibiotic-resistance genes (ARGs), imposing the most critical health threats to humans and the environment. Continuous technological advances have led to constant improvements and expansions of molecular methods, such as conventional end-point PCR, DNA microarray, real-time quantitative PCR (qPCR), multiplex qPCR (mqPCR), loop-mediated isothermal amplification (LAMP), digital droplet PCR (ddPCR), and high-throughput next-generation DNA sequencing (HT-NGS). These state-of-the-art molecular approaches largely facilitate the surveillance of microbial water quality in diverse aquatic systems and wastewater. This review provides an up-to-date overview of the advancement of the key molecular tools frequently employed for microbial water quality assessment, with future perspectives on their applications.

A population-level statistic for assessing Mendelian behavior of genotyping-by-sequencing data from highly duplicated genomes.

BACKGROUND: Given the economic and environmental importance of allopolyploids and other species with highly duplicated genomes, there is a need for methods to distinguish paralogs, i.e. duplicate sequences within a genome, from Mendelian loci, i.e. single copy sequences that pair at meiosis. The ratio of observed to expected heterozygosity is an effective tool for filtering loci but requires genotyping to be performed first at a high computational cost, whereas counting the number of sequence tags detected per genotype is computationally quick but very ineffective in inbred or polyploid populations. Therefore, new methods are needed for filtering paralogs. RESULTS: We introduce a novel statistic, Hind/HE, that uses the probability that two reads sampled from a genotype will belong to different alleles, instead of observed heterozygosity. The expected value of Hind/HE is the same across all loci in a dataset, regardless of read depth or allele frequency. In contrast to methods based on observed heterozygosity, it can be estimated and used for filtering loci prior to genotype calling. In addition to filtering paralogs, it can be used to filter loci with null alleles or high overdispersion, and identify individuals with unexpected ploidy and hybrid status. We demonstrate that the statistic is useful at read depths as low as five to 10, well below the depth needed for accurate genotype calling in polyploid and outcrossing species. CONCLUSIONS: Our methodology for estimating Hind/HE across loci and individuals, as well as determining reasonable thresholds for filtering loci, is implemented in polyRAD v1.6, available at https://github.com/lvclark/polyRAD . In large sequencing datasets, we anticipate that the ability to filter markers and identify problematic individuals prior to genotype calling will save researchers considerable computational time.

Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.

Binding affinity is one of the primary determinants of antibody function. Here, we provide a protocol for simple and rapid affinity maturation of single-domain antibodies (sdAbs) using tandem phage display selection and next-generation DNA sequencing. The sequence of a model camelid sdAb directed against Clostridioides difficile toxin A (A26.8) was diversified using either random or site-saturation mutagenesis and cloned into a phagemid vector upstream of gene 3. The resulting phage-displayed sdAb libraries were panned against C. difficile toxin A and the panning outputs interrogated using Illumina MiSeq sequencing. Through bioinformatic analyses, we were able to identify individual affinity-enhancing amino acid substitutions in the sdAb complementarity-determining regions that, when combined, resulted in affinity improvements of approximately 10-fold. The advantages of this method are that it does not require extensive screening and characterization of individual clones, nor structural information on the mechanism of the sdAb:antigen interaction.

The Bacterial and Fungal Microbiota of the Mexican Rubiaceae Family Medicinal Plant Bouvardia ternifolia.

Bouvardia ternifolia is a medicinal plant considered a source of therapeutic compounds, like the antitumoral cyclohexapeptide bouvardin. It is known that large number of secondary metabolites produced by plants results from the interaction of the host and adjacent or embedded microorganisms. Using high-throughput DNA sequencing of V3-16S and V5-18S ribosomal gene libraries, we characterized the endophytic, endophytic + epiphyte bacterial, and fungal communities associated to flowers, leaves, stems, and roots, as well as the rhizosphere. The Proteobacteria (average 80.7%) and Actinobacteria (average 14.7%) were the most abundant bacterial phyla, while Leotiomycetes (average 54.8%) and Dothideomycetes (average 27.4%) were the most abundant fungal classes. Differential abundance for the bacterial endophyte group showed a predominance of Erwinia, Propionibacterium, and Microbacterium genera, while Sclerotinia, Coccomyces, and Calycina genera predominated for fungi. The predictive metagenome analysis for bacteria showed significative abundance of pathways for secondary metabolite production, while a FUNguild analysis revealed the presence of pathotroph, symbiotroph, and saprotrophs in the fungal community. Intra and inter copresence and mutual exclusion interactions were identified for bacterial and fungal kingdoms in the endophyte communities. This work provides a description of the diversity and composition of bacterial and fungal microorganisms living in flowers, leaves, stems, roots, and the rhizosphere of this medicinal plant; thus, it paves the way towards an integral understanding in the production of therapeutic metabolites.

Systems Biology of the Vasopressin V2 Receptor: New Tools for Discovery of Molecular Actions of a GPCR.

Systems biology can be defined as the study of a biological process in which all of the relevant components are investigated together in parallel to discover the mechanism. Although the approach is not new, it has come to the forefront as a result of genome sequencing projects completed in the first few years of the current century. It has elements of large-scale data acquisition (chiefly next-generation sequencing-based methods and protein mass spectrometry) and large-scale data analysis (big data integration and Bayesian modeling). Here we discuss these methodologies and show how they can be applied to understand the downstream effects of GPCR signaling, specifically looking at how the neurohypophyseal peptide hormone vasopressin, working through the V2 receptor and PKA activation, regulates the water channel aquaporin-2. The emerging picture provides a detailedframework for understanding the molecular mechanisms involved in water balance disorders, pointing the way to improved treatment of both polyuric disorders and water-retention disorders causing dilutional hyponatremia.

Lactococcus garvieae Periprosthetic Joint Infection in a Cattle Rancher with a Total Knee Arthroplasty: A Novel Reservoir Transmission.

Introduction: Periprosthetic joint infection (PJI) remains a challenging complication of joint replacement surgery. With the more frequent use of immune modifying drugs and dietary changes in human populations, the resultant blunting of immune defenses allows for infections with less common organisms. Case Report: Lactococcus garvieae is an anaerobic, gram-positive coccus with reservoirs in fish and domesticated farm animals. Only two prior cases of PJI due to L. garvieae have been reported, both with reported marine transmission. We report a case of L. garvieae associated PJI in a cattle rancher with the first reported case of transmission from a bovine reservoir. The PJI was associated with intra-articular rice body formation, and the diagnosis confirmed with the aid of next generation DNA sequencing. A successful two stage exchange was performed. We propose a novel transmission mechanism with microbe entry via direct hematogenous inoculation during the patient's duties as a rancher. Conclusion: When an unusual organism is detected in a PJI, the treatment team should research the host reservoir(s) of the organism and correlate with the patient's exposure risk. While contamination of cultures is possible, a thorough investigation should be performed prior to that assumption. This reinforces the basic concept that a careful history remains vital when treating an unusual infection presentation. Next generation DNA sequencing is a useful confirmatory tool in establishing the offending organism. Lastly, the identification of rice bodies should raise suspicion for infection. Although not always associated with infection, efforts should be redoubled to identify or rule out a causative micro-organism(s).