The Role of Proteases and Serpin Protease Inhibitors in β-Cell Biology and Diabetes

Regulation of the equilibrium between proteases and their inhibitors is fundamental to health maintenance. Consequently, developing a means of targeting protease activity to promote tissue regeneration and inhibit inflammation may offer a new strategy in therapy development for diabetes and other diseases. Specifically, recent efforts have focused on serine protease inhibitors, known as serpins, as potential therapeutic targets. The serpin protein family comprises a broad range of protease inhibitors, which are categorized into 16 clades that are all extracellular, with the exception of Clade B, which controls mostly intracellular proteases, including both serine- and papain-like cysteine proteases. This review discusses the most salient, and sometimes opposing, views that either inhibition or augmentation of protease activity can bring about positive outcomes in pancreatic islet biology and inflammation. These potential discrepancies can be reconciled at the molecular level as specific proteases and serpins regulate distinct signaling pathways, thereby playing equally distinct roles in health and disease development.

Chromosome Evolution in the Genus Partamona (Apidae: Meliponini), with Comments on B Chromosome Origin

The genus <i>Partamona</i> includes 33 species of stingless bees, of which 11 were studied cytogenetically. The main goal of this study was to propose a hypothesis about chromosomal evolution in <i>Partamona</i> by combining molecular and cytogenetic data. Cytogenetic analyses were performed on 3 <i>Partamona</i> species. In addition, the molecular phylogeny included mitochondrial sequences of 11 species. Although the diploid number was constant within the genus, 2n = 34, B chromosomes were reported in 7 species. Cytogenetic data showed karyotypic variations related to chromosome morphology and the amount and distribution of heterochromatin and repetitive DNA. The molecular phylogenetic reconstruction corroborated the monophyly of the genus and separated the 2 clades (A and B). This separation was also observed in the cytogenetic data, in which species within each clade shared most of the cytogenetic characteristics. Furthermore, our data suggested that the B chromosome in the genus <i>Partamona</i> likely originated from a common ancestor of the species that have it in clade B and, through interspecific hybridization, it appeared only in <i>Partamona rustica</i> from clade A. Based on the above, <i>Partamona</i> is an interesting genus for further investigations using molecular mapping of B chromosomes as well as for broadening phylogenetic data.

1008. Presence of Antibody Dependent Cell Cytotoxicity (ADCC) Functional Antibodies that Target a Complex Gp41 Epitope Correlates with Long-term Non-progression and ADCC is Maintained with Mutants Using Germline Heavy Chain Variable Gene Sequence of VH1-02 Gene

Background Recent data supports that improved qualitative antibody responses correlate with elite controllers (EC) of HIV. As ADCC has been associated with protection in vaccine studies, thorough exploration of antibodies that facilitate ADCC is warranted. In studies on monoclonal antibodies from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated antibodies against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing antibodies against HIV, members of these antibodies, termed group 76C antibodies, did not exhibit broad neutralization. Methods Our goal was to characterize the non-neutralizing functions of antibodies of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these antibodies by comparison to their predicted common ancestor. Serum samples were obtained from HIV+ clinical groups: EC, LTNP, stable CD4 counts on therapy, and those off therapy. Results In antibody/serum competition assays, comparison to VRC01 which also uses VH1-02, showed that antibodies targeting the 76C group epitope were enriched in LTNPs. We then show recombinant antibodies of 76C members 6F5 and 6F11 both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc also shows comparable ADCC to 6F5 and 6F11 on both clade B and C constructs. Common ancestor antibodies maintain function and these types of antibodies correlate to a non-progressive clinical state. (A) Serum from long-term non-progressors (LTNPs) compared to serum from a group of HIV infected with lower CD4 levels as a control for viral load were used to compete against biotinylated CD4 binding site (VRC01) and 76C Gp41 conformational epitope (6F11) targeting antibodies. Serum dilutions were chosen to align means near 50%. Means with 95% confidence intervals are shown. (B) Monoclonal antibody 76Canc was created using the germline sequence of the heavy chain variable region with the CDR3 and light chain of 76C member. Antibody dependent cell cytotoxicity flow cytometric based assays were performed using gp41 proteins from clade B (MN) and clade C (ZA1197). Conclusion Certain antibodies present early on in infection may contribute to overall clinical course. Variable gene germline sequences that support functional activity against HIV could be targeted in vaccine regimens. Disclosures All Authors: No reported disclosures

SERPINB6 expression associates with survival in triple negative breast cancer.

We mined published microarray data (1) to understand the most significant gene expression differences in the tumors of triple negative breast cancer patients based on survival following treatment: dead or alive. We observed significant transcriptome-wide differential expression of serpin peptidase inhibitor, clade B (ovalbumin), member 6, encoded by SERPINB6 when comparing the primary tumors of triple negative breast cancer patients dead or alive. Importantly, SERPINB6 expression was correlated with post-progression survival in basal subtype breast cancer, a molecular subtype sharing significant overlap with triple negative breast cancer. SERPINB6 may be of relevance as a biomarker or as a molecule of interest in understanding the etiology or progression of triple negative breast cancer.

SERPINB9 expression associates with survival in triple negative breast cancer.

We mined published microarray data (1) to understand the most significant gene expression differences in the tumors of triple negative breast cancer patients based on survival following treatment: dead or alive. We observed significant transcriptome-wide differential expression of serpin peptidase inhibitor, clade B (ovalbumin), member 9, encoded by SERPINB9 when comparing the primary tumors of triple negative breast cancer patients dead or alive. Importantly, SERPINB9 expression was correlated with recurrence-free survival in basal subtype breast cancer, a molecular subtype sharing significant overlap with triple negative breast cancer. SERPINB9 may be of relevance as a biomarker or as a molecule of interest in understanding the etiology or progression of triple negative breast cancer.

The unmutated common ancestor of Gp41 conformational epitope targeting variable heavy chain 1-02 utilizing antibodies maintains antibody dependent cell cytotoxicity

In studies on monoclonal Abs (mAbs) from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated Abs against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing Abs against HIV, members of these Abs, termed group 76C Abs, did not exhibit broad neutralization.<br />Because of the excessive mutations and use of VH1-02, our goal was to characterize the non-neutralizing functions of Abs of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these Abs by comparison to their predicted common ancestor. Serum competition assays showed group 76C Abs were enriched in LTNPs, in comparison to VRC-01. Specific group 76C clones 6F5 and 6F11, expressed as recombinant Abs, both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. Competition with group 76C recombinant Ab 6F5 confirms 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc demonstrates comparable ADCC to 6F5 and 6F11 when targeting both clade B and C HIV constructs. The functional capability of Abs utilizing germline VH1-02 has implications for disease control and vaccine development.

Subtype Differences in the Interaction of HIV-1 Matrix with Calmodulin: Implications for Biological Functions

The HIV-1 Gag polyprotein plays essential roles during the late stage of the HIV-1 replication cycle, and has recently been identified as a promising therapeutic target. The N-terminal portion of the HIV-1 Gag polyprotein encodes the myristoylated matrix (MA) protein, which functions in the trafficking of the structural proteins to the plasma membrane (PM) and facilitation of envelope incorporation into budding virus. Numerous host cell proteins interact with the MA portion of the Gag polyprotein during this process. One such factor is the ubiquitous calcium-binding protein calmodulin (CaM), which interacts preferentially with myristoylated proteins, thereby regulating cell physiology. The exact role of this interaction is poorly understood to date. Atomic resolution structures revealed the nature of the CaM-MA interaction for clade B isolates. In this study, we expanded our knowledge and characterized biophysically and computationally the CaM interaction with MA from other HIV-1 clades and discovered differences in the CaM recognition as compared to the prototypical clade B MA., with significant alterations in the interaction with the MA protein from clade C. Structural investigation and in silico mutational analysis revealed that HIV-1 MA protein from clade C, which is responsible for the majority of global HIV-1 infections, interacts with lower affinity and altered kinetics as compared to the canonical clade B. This finding may have implications for additional altered interaction networks as compared to the well-studied clade B. Our analysis highlights the importance of expanding investigations of virus-host cell factor interaction networks to other HIV-1 clades.

Genome-based studies indicate that the Enterococcus faecium Clade B strains belong to Enterococcus lactis species and lack of the hospital infection associated markers

Enterococcus lactis and the heterotypic synonym Enterococcus xinjiangensis from dairy origin have recently been identified as a novel species based on 16S rRNA gene sequence analysis. Enterococcus faecium type strain NCTC 7171T was used as the reference genome for determining E. lactis and E. faecium to be separate species. However, this taxonomic classification did not consider the diverse lineages of E. faecium , and the double nature of hospital-associated (clade A) and community-associated (clade B) isolates. Here, we investigated the taxonomic relationship among isolates of E. faecium of different origins and E. lactis , using a genome-based approach. Additional to 16S rRNA gene sequence analysis, we estimated the relatedness among strains and species using phylogenomics based on the core pangenome, multilocus sequence typing, the average nucleotide identity and digital DNA–DNA hybridization. Moreover, following the available safety assessment schemes, we evaluated the virulence profile and the ampicillin resistance of E. lactis and E. faecium clade B strains. Our results confirmed the genetic and evolutionary differences between clade A and the intertwined clade B and E. lactis group. We also confirmed the absence in these strains of virulence gene markers IS16, hylEfm and esp and the lack of the PBP5 allelic profile associated with ampicillin resistance. Taken together, our findings support the reassignment of the strains of E. faecium clade B as E. lactis .

A cross-sectional survey of hard ticks and molecular characterization of Rhipicephalus microplus parasitizing domestic animals of Khyber Pakhtunkhwa, Pakistan

Background In tropical and subtropical countries, tick infestation causes major public health problems and considerable financial losses to the livestock industry. This study was aimed to assess the species composition of richness and analyze the phylogeny of Rhipicephalus microplus in the District Bannu of Khyber Pakhtunkhwa, Pakistan. Methods Collected ticks were identified morphologically and DNA extracted from R. microplus was amplified and subjected to sequencing. Results A total of 3,600 animals were examined among them 1,494 animals were found to be infested with ticks, including 669 cows, 476 buffaloes, 163 goats, and 186 sheep (p = 0.001). Tick infestation was significantly high (43.58%) in animals of age group (<1 year) (p-value = 0.027). Female animals were more (44.05%) infested with ticks than males (34.43%) (p = 0.001). The intensity of infestation was significantly higher in summer (77.49%) (p = 0.001). A total of 5,557 ticks were collected comprising three genera and six species. R. microplus was predominantly prevalent (n = 1,474; 26.52%), followed by Rhipicephalus annulatus (n = 1,215; 21.86%), Hyalomma anatolicum (n = 1,139; 20.49%), Hyalomma marginatum (n = 1,086; 19.54%), and Rhipicephalus turanicus (n = 761; 13.69%), while the least common was Haemaphysalis aciculifer (n = 80; 1.43%) (p = 0.001). Morphologically identified R. microplus species were also analyzed genetically by using two genetic markers 16S ribosomal RNA (16S rRNA) and internal transcribed spacer 2 (ITS2) genes. The phylogenetic study revealed that R. microplus is genetically diversified and clustered in clade B with R. microplus species from China, India, and Pakistan. Conclusion Ticks infestation was significantly correlated with various factors including age, sex, season, and animal type. R. microplus genetically resembled species reported from India and China. However, major knowledge gaps concerning various species of ticks exist and many areas are still unexplored in Pakistan. Therefore, it is necessary to explore the epidemiological and molecular aspects of various tick species in other regions of southern Khyber Pakhtunkhwa.