Development of a ‘genetic signature of environmental lead exposure’ in wild Peromyscus using combinatorial data from cDNA microarrays and blood metabolites

A study of environmental toxicology must be able to identify the biomarkers of one or a few related effectors among a myriad of toxicants affecting diverse biological factors in one or more organisms residing in a contaminated environment and at present, no available technique meets this unique requirement. Lead (Pb) toxicity remains an important environmental toxicological problem on a global scale because of the prevalence of lead exposure to adults and children and its impact on public health (Lanphear, 2007; Hu, et al., 2007). In addition, lead exposure equally affects organisms in natural ecosystems, since lead is ubiquitously found in the environment both as a contaminant from man made materials and natural sources. In addition, the lead is not metabolized in the body, which makes it ideal for the preliminary detection of biomarkers for environmental toxicants. The fundamental goal of my research study was to identify and assess the effectiveness of DNA microarray technology for detection of the biological impact of environmental lead (Pb). I worked it out by identifying correlation between gene expression data from the DNA microarray and conventional toxicological data, such as change of blood Pb and heme concentrations, resulting from environmental lead exposure. I linked the gene expression profiles to the well known lead inducible biochemical processes, to discover ‘genetic signature of lead exposure’ in the liver and spleen of laboratory Peromyscus maniculatus and Rattus norvagicus, two widely available rodents. I used this laboratory data to develop the genetic signature of environmental lead in the wild Peromyscus maniculatus collected from contaminated field. I found that lead significantly affected expression of the nuclear genes of NADH dehydrogenase and cytochrome oxidase of mitochondrial electron transport system, acyl CoA metabolism, calmodulin, a multifunctional Ca-binding protein, and heat shock protein-1 (90KDa) from stress pathway. However, in these rodents lead induced anemia without affecting the expression of genes in hemoglobin metabolism.Ph.D., Environmental Toxicology -- Drexel University, 200

CLINICAL INSIGHT ON PATTERNS OF CARE AND PROGNOSTIC FACTORS IN ADULT HIGH GRADE GLIOMA: EXPERIENCE FROM A TERTIARY CANCER HOSPITAL FROM EASTERN INDIA

Objective: The Central Nervous System Tumors account for 2.4% of all malignancies in India, but are associated with high mortality in high-grade tumors which result in poor death-adjusted life years. This study focuses on patterns of care and prognostic factors of adult high-grade glioma to explore the unaddressed nuances in treating such patients. Methods: It was a retrospective single institutional study from June 2018 to July 2021 with an age group between 16 to 70 years. All histopathologically or clinicoradiologically proven cases of high-grade (World Health Organization Grades III and IV) gliomas were assessed. Defaulters and recurrent glioma at presentation were excluded from the analysis. Baseline characteristics were analyzed by Chi-square and unpaired t-test, and the Kaplan– Meir test was used for survival analysis. p<0.05 was considered significant. Results: 41 patients were accrued for final analysis with a median follow-up period of 18 months. The most common histology was Astrocytoma, followed by Glioblastoma with a female preponderance. The Frontal and Temporal lobe was the predominant site in the study population. A majority (82%) of the patients underwent maximal safe resection followed by chemoradiation therapy (63.4%). Median progression free survival was 24 months and 8 months for Grades III and IV gliomas, respectively. The median overall survival for Grade IV gliomas was 7 months. Conclusion: Resection status, Grade IV, IDH and 1p19q codeletion status were significant prognostic factors, while intensity modulated radiotherapy showed better dosimetry. More prospective randomized studies with larger sample sizes and longer follow-ups are required for validation and drafting an outcome nomogram

Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes

We investigated the intracellular route of Salmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonella-containing phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, live Salmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (Mr17,000) was detected by 30 min on the dead Salmonella-containing phagosomes. Morphological analysis also revealed that after uptake by macrophages, the dead Salmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment

Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

BACKGROUND: SPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them. RESULTS: In large (25 mm) wounds, SPARC-null mice showed a significant delay in healing as compared to wild-type mice (31 days versus 24 days). Granulation tissue formation and extracellular matrix protein production were delayed in small 6 mm SPARC-null wounds initially but were resolved by day 6. In in vitro wound-healing assays, while wild-type primary dermal fibroblasts showed essentially complete wound closure at 11 hours, wound closure of SPARC-null cells was incomplete even at 31 hours. Addition of purified SPARC restored the normal time course of wound closure. Treatment of SPARC-null cells with mitomycin C to analyze cell migration without cell proliferation showed that wound repair remained incomplete after 31 hours. Cell proliferation as measured by (3)H-thymidine incorporation and collagen gel contraction by SPARC-null cells were not compromised. CONCLUSIONS: A significant delay in healing large excisional wounds and setback in granulation tissue formation and extracellular matrix protein production in small wounds establish that SPARC is required for granulation tissue formation during normal repair of skin wounds in mice. A defect in wound closure in vitro indicates that SPARC regulates cell migration. We conclude that SPARC plays a role in wound repair by promoting fibroblast migration and thus granulation tissue formation

Oligonucleotides tethered to a short polyguanylic acid stretch are targeted to macrophages: enhanced antiviral activity of a vesicular stomatitis virus-specific antisense oligonucleotide

The poor membrane permeability of oligonucleotides is one of the major problems of antisense technology. Here we report the construction of designer oligonucleotides for targeted delivery to macrophages. The oligonucleotides tethered to a 10-mer poly(G) sequence at their 3' ends were recognized by scavenger receptors on macrophages and were taken up about 8- to 10-fold as efficiently as those oligonucleotides that either lacked a poly(G) tail or that contained a 10-mer poly(C) tail instead of the poly(G) tail. The enhanced uptake of poly(G) constructs was inhibited in the presence of poly(G) and other known ligands of the scavenger receptor. The bioefficacy of poly(G)-mediated targeting of antisense oligonucleotides (ANS) was demonstrated by using vesicular stomatitis virus (VSV) as a model system. The ability of ANS directed against the translation initiation site of N protein mRNA of VSV to inhibit virus replication was assessed. The ANS with the 10-mer poly(G) sequences (ANS-G) brought about significant inhibition of VSV replication in J774E cells (a murine monocyte/macrophage cell line) and Chinese hamster ovary (CHO) cell transfectants expressing scavenger receptors. The ANS lacking a 10-mer poly(G) stretch were ineffective. The inhibition of VSV replication due to ANS-G was completely abrogated in the presence of 10-mer poly(G), indicating that the antisense effect of the ANS-G molecule was a consequence of scavenger receptor-mediated enhanced uptake. Importantly, antisense molecules linked exclusively by natural phosphodiester bonds were as bioeffective as those synthesized with a mixed backbone of phosphodiester and phosphorothioate. Taken together, these results suggest that macrophage-directed designer ANS against infective agents may simply be obtained by adding a short stretch of guanylic acid sequence to the desired specific ANS during solid-phase synthesis. This nucleic acid-based strategy, which utilizes homogeneous preparation of ANS, may find applications in directed manipulation of macrophage metabolism for a variety of purposes as well as in therapy of a broad spectrum of macrophage-related disorders amenable to the antisense approach

Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome

To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPγS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, α-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPγS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of α-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes

Diverting intracellular trafficking of Salmonella to the lysosome through activation of the late endocytic Rab7 by intracellular delivery of muramyl dipeptide

Previously, we showed that live Salmonella-containing phagosomes (LSP) recruit early acting Rab5 and promote fusion with early endosomes, thus avoiding transport to the lysosomes. Therefore, live Salmonella survive in a specialized compartment. Here we show that scavenger-receptor-mediated intracellular delivery of muramyl dipeptide (MDP) to macrophages leads to efficient killing of Salmonella both in vitro and in vivo. To understand the intracellular trafficking modulation of Salmonella by delivery of MDP, we investigated the levels of endocytic Rab proteins, which are the major regulators of vesicular transport. Western blot analysis reveals reduced Rab5 and enhanced Rab7 content in the maleylated bovine serum albumin-MDP (MBSA-MDP)-treated cells. The reduced content of Rab5 in the treated cells and on phagosomes inhibits the fusion of Salmonella-containing phagosomes with early endosomes, and the enhanced Rab7 content in these cells facilitated targeting of LSP to lysosomes, which contain cathepsin D and vacuolar ATPase, for killing. In vitro reconstitution of lysosomal transport demonstrated that a reduced content of Rab5 and an enhanced level of Rab7 in MBSA-MDP-treated cells is primarily responsible for targeting Salmonella to lysosomes. Intracellular delivery of MDP thus offers a general strategy against macrophage-associated infections caused by intracellular pathogens that survive in the host cell by resisting transport to lysosomes

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-ΔC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of 125I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-ΔC antibody and Ld-HbR-ΔC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis

Oncocytic lesion of parotid gland: A dilemma for cytopathologists

Oncocytes are epithelial cells with abundant, granular, eosinophilic cytoplasm due to presence of numerous large mitochondria of varied sizes. The presence of oncocytes in salivary glands can occur in a variety of conditions. Here, we present a rare case of a 68 year old male patient who presented with a 6 cm diameter swelling in the right parotid region. A fine needle aspiration cytology done from the lesion showed a cellular oncocytic lesion. A possibility of oncocytoma was entertained. Histopathology of the mass showed a rare entity called diffuse hyperplastic oncocytosis. Originally believed to be a metaplastic process, oncocytes can occur in various lesions ranging from hyperplastic conditions to malignant neoplasms. However, diagnosis on cytological smears can be very challenging for the cytopathologist