Prevalence and Recovery of Microorganisms from Containers used for the Collection of Forensic Biological Samples

Background: Microbes play a significant role in the degradation of biological evidence collected for forensic analysis. The present study is aimed to isolate and identify the microbes present inside the empty container used for the biological evidence collection. Methods: Bacterial isolation from the selected containers was done by cotton swab over the inner surface of the containers. Streaking was done on the surface of the three different culture plates as a Blood agar plate, Nutrient plate and MacConkey plate. The plates were placed in an incubator shaker at 37ºC for 48 hours. The colonies grown on the surface of the media were counted on and used for further study. Various biochemical assays were performed to characterize isolated bacteria. Results: Staining results suggested that the presence of Gram-positive stain (Staphylococcus, Bacillus, Corynebacterium, Clostridium) and Gram negative stain (E. coli, Enterobacteriaceae, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Bdellovibrio, Acetic acid bacteria). The Catalase and Coagulase test suggested the presence of Staphylococcus aureus, S. epidermis and S. sapropyticus. Moreover, the indole test suggested the presence of Citrobacter koseri, Kebsiella oxytoca, Proteus vulgaris etc. Some of the bacteria were urea metabolizing, including Proteus spp, Helicobacter pylori, Cryptococcus spp, Corynebacterium spp. Conclusion: This study recommends that there should be proper maintenance of the chain of custody from the collection to analysis so that evidence properly prevents degradation or contamination in the biological evidence. Extra care is needed for the collection and packing of biological evidence from the crime scene. Moreover, the collection containers, if left wide open, lead to contamination and degradation of biological evidence.

Structural arrangement of the VH and VL domains in the COBRA™ T-cell engaging single chain diabody

Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.

Molecular architecture of nucleosome remodeling and deacetylase sub-complexes by integrative structure determination

The Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin-modifying assembly that regulates gene expression and DNA damage repair. Despite its importance, limited structural information is available on the complex and a detailed understanding of its mechanism is lacking. We investigated the molecular architecture of three NuRD sub-complexes: MTA1-HDAC1-RBBP4 (MHR), MTA1N-HDAC1-MBD3GATAD2CC (MHM), and MTA1-HDAC1-RBBP4-MBD3-GATAD2 (NuDe) using Bayesian integrative structure determination with IMP (Integrative Modeling Platform), drawing on information from SEC-MALLS, DIA-MS, XLMS, negative stain EM, X-ray crystallography, NMR spectroscopy, secondary structure and homology predictions. The structures were corroborated by independent cryo-EM maps, biochemical assays, and known cancer-associated mutations. Our integrative structure of the 2:2:2 MHM complex shows asymmetric binding of MBD3, whereas our structure of the NuDe complex shows MBD3 localized precisely to a single position distant from the MTA1 dimerization interface. Our models suggest a possible mechanism by which asymmetry is introduced in NuRD, and indicate three previously unrecognized subunit interfaces in NuDe: HDAC1C-MTA1BAH, MTA1BAH-MBD3, and HDAC160-100-MBD3. We observed that a significant number of cancer-associated mutations mapped to protein-protein interfaces in NuDe. Our approach also allows us to localize regions of unknown structure, such as HDAC1C and MBD3IDR, thereby resulting in the most complete structural characterization of these NuRD sub-complexes so far.

Pentameric assembly of the Kv2.1 tetramerization domain.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristic of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily missing a representative structure of the T1 domain is the Kv2 family. We used X-ray crystallography to solve the structure of the human Kv2.1 T1 domain. The structure is similar to other T1 domains but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer as determined with in-line SEC-MALS-SAXS and negative stain EM. The Kv2.1 T1-T1 interface involves electrostatic interactions including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. We show that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains which may reflect the versatility of Kv2.1, the only Kv subfamily that can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

Study the role of c-reactive protein and thrombocytopenia in diagnosing early neonatal sepsis

Background: In clinical practice, early detection of neonatal sepsis remains difficult. Neonatal sepsis diagnosis is a challenge because of its non-specific presentation and the low sensitivity of the time-consuming bacterial cultures. So, many sepsis markers, like C-reactive protein (CRP) and platelet count, are emerging to improve its diagnosis. The aim of the study was to evaluate the role of CRP and platelet count in early neonatal sepsisMethods: This study was conducted in the department of SNCU in Virudhunagar Government Headquarters hospital, 50 neonates with culture-confirmed sepsis were included. Before antibiotic treatment, neonates CRP and platelet count, and other inflammatory markers were assessed.Results: In 50 newborns, culture was positive in 22 cases, CRP was positive in 24 cases, and thrombocytopenia in 18 cases. In 22 culture-positive cases, 19 cases are CRP positive, and 17 cases had thrombocytopenia. In 17 culture-positive thrombocytopenia cases, 16 cases are Gram-negative stain, 1 case is Gram-positive stain.Conclusions: CRP and Platelet can be helpful in the future diagnosis of neonatal sepsis, despite being promising and convenient markers for neonatal sepsis.

Structural basis of LAIR1 targeting by polymorphic Plasmodium RIFINs

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

Protection of the prodomain 1-helix correlates with latency in the transforming growth factor- family

The 33 members of the transforming growth factor beta (TGF-) family are fundamentally important for organismal development and homeostasis. Family members are synthesized and secreted as pro-complexes of prodomains that are non-covalently bound to the growth factor (GF). The pro-complexes of some members are latent and require activation steps to release the GF for signaling. Why some members are latent while others are non-latent is incompletely understood, but crystal structures and hydrogen-deuterium exchange (HDX) of four family members have begun to unravel how latency is regulated. Here, we extend this understanding by comparing pro-complex conformation in negative stain EM (nsEM) and HDX of ActA, BMP7, BMP9, BMP10, GDF8, TGF-1, and TGF-2. nsEM revealed that family members varied in either adopting cross-armed, open-armed, or V-armed configurations. Latency was achieved in both cross-armed and V-armed but not open-armed conformations. HDX revealed remarkably varying patterns of exchange between family members, consistent with large prodomain sequence divergence. We observed a strong correlation between latency and protection of the prodomain 1-helix from exchange, which in latent members coincided with greater buried surface area of the 1-helix and more hydrogen and cation-pi bonds from the prodomain fastener and GF to the 1-helix. Strong sequence conservation of the 1-helix and fastener only in latent members suggests that similar interactions are conserved and sufficient to confer latency. Moreover, most members exhibited rapid exchange in the unstructured association region at the prodomain N-terminus, highlighting their availability for interacting with factors that may regulate latency and extracellular storage.

Scaling Concepts in Serpin Polymer Physics

α1-Antitrypsin is a protease inhibitor belonging to the serpin family. Serpin polymerisation is at the core of a class of genetic conformational diseases called serpinopathies. These polymers are known to be unbranched, flexible, and heterogeneous in size with a beads-on-a-string appearance viewed by negative stain electron microscopy. Here, we use atomic force microscopy and time-lapse dynamic light scattering to measure polymer size and shape for wild-type (M) and Glu342→Lys (Z) α1-antitrypsin, the most common variant that leads to severe pathological deficiency. Our data for small polymers deposited onto mica and in solution reveal a power law relation between the polymer size, namely the end-to-end distance or the hydrodynamic radius, and the polymer mass, proportional to the contour length. We use the scaling concepts of polymer physics to assess that α1-antitrypsin polymers are random linear chains with a low persistence length.

Identification of recombinant Fabs for structural and functional characterization of HIV-host factor complexes

Viral infection and pathogenesis is mediated by host protein—viral protein complexes that are important targets for therapeutic intervention as they are potentially less prone to development of drug resistance. We have identified human, recombinant antibodies (Fabs) from a phage display library that bind to three HIV-host complexes. We used these Fabs to 1) stabilize the complexes for structural studies; and 2) facilitate characterization of the function of these complexes. Specifically, we generated recombinant Fabs to Vif-CBF-β-ELOB-ELOC (VCBC); ESCRT-I complex and AP2-complex. For each complex we measured binding affinities with KD values of Fabs ranging from 12–419 nM and performed negative stain electron microscopy (nsEM) to obtain low-resolution structures of the HIV-Fab complexes. Select Fabs were converted to scFvs to allow them to fold intracellularly and perturb HIV-host protein complex assembly without affecting other pathways. To identify these recombinant Fabs, we developed a rapid screening pipeline that uses quantitative ELISAs and nsEM to establish whether the Fabs have overlapping or independent epitopes. This pipeline approach is generally applicable to other particularly challenging antigens that are refractory to immunization strategies for antibody generation including multi-protein complexes providing specific, reproducible, and renewable antibody reagents for research and clinical applications. The curated antibodies described here are available to the scientific community for further structural and functional studies on these critical HIV host-factor proteins.