MRI-Based Cell Tracking of OATP-Expressing Cell Transplants by Pre-Labeling with Gd-EOB-DTPA.

PURPOSE: A critical step in cell-based therapies is determining the exact position of transplanted cells immediately post-transplant. Here, we devised a method to detect cell transplants immediately post-transplant, using a clinical gadolinium-based contrast agent. These cells were detected as hyperintense signals using a clinically familiar T1-weighted MRI protocol. PROCEDURES: HEK293 cells were stably transduced to express human OATP1B3, a hepatic organic anion transporting polypeptide that transports Gd-EOB-DTPA into cells that express the transporters, the intracellular accumulation of which cells causes signal enhancement on T1-weighted MRI. Cells were pre-labeled prior to injection in media containing Gd-EOB-DTPA for MRI evaluation and indocyanine green for cryofluorescence tomography validation. Labeled cells were injected into chicken hearts, in vitro, after which MRI and cryofluorescence tomography were performed in sequence. RESULTS: OATP1B3-expressing cells had substantially reduced T1 following labeling with Gd-EOB-DTPA in culture. Following their implantation into chicken heart, these cells were robustly identified in T1-weighted MRI, with image-derived injection volumes of cells commensurate with intended injection volumes. Cryofluorescence tomography showed that the areas of signal enhancement in MRI overlapped with areas of indocyanine green signal, indicating that MRI signal enhancement was due to the transplanted cells. CONCLUSIONS: OATP1B3-expressing cells can be pre-labeled with Gd-EOB-DTPA prior to injection into tissue, affording the use of clinically familiar T1-weighted MRI to robustly detect cell transplants immediately after transplant. This procedure is easily generalizable and has potential advantages over the use of iron oxide based cell labeling agents and imaging procedures.

First Report of Anopheles annularis s.l., An. maculatus s.s., and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l., An. hyrcanus s.l., An. splendidus, and An. vagus were morphologically identified. For species like An. annularis s.l., An. minimus s.s., An. culicifacies s.l., and An. maculatus s.s., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l., An. maculatus s.s., An. culicifacies s.l., An. vagus, and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis. The major Anopheles species were An. annularis s.l., An. culicifacies s.l., and An. maculatus s.s., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s., An. culicifacies s.l., and An. annularis s.l. as significant vectors in the Alipurduar region.

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

During gene regulation, DNA accessibility is thought to limit the availability of transcription factor (TF) binding sites, while TFs can increase DNA accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events in the modulation of gene expression remain unknown for the vast majority of genes. We utilized deeply sequenced ATAC-Seq data and site-specific knock-in reporter genes to investigate the relationship between the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of Cebpa during macrophage-neutrophil differentiation. While the enhancers upregulate reporter expression during the earliest stages of differentiation, there is little corresponding increase in their total accessibility. Conversely, total accessibility peaks during the last stages of differentiation without any increase in enhancer activity. The accessibility of positions neighboring C/EBP-family TF binding sites, which indicates TF occupancy, does increase significantly during early differentiation, showing that the early upregulation of enhancer activity is driven by TF binding. These results imply that a generalized increase in DNA accessibility is not sufficient, and binding by enhancer-specific TFs is necessary, for the upregulation of gene expression. Additionally, high-coverage ATAC-Seq combined with time-series expression data can infer the sequence of regulatory events at binding-site resolution.

The contributions of DNA accessibility and transcription factor occupancy to enhancer activity during cellular differentiation.

The upregulation of gene expression by enhancers depends upon the interplay between the binding of sequence-specific transcription factors (TFs) and DNA accessibility. DNA accessibility is thought to limit the ability of TFs to bind to their sites, while TFs can increase accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events underlying the modulation of gene expression during cellular differentiation remain unknown for the vast majority of genes. We investigated the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of an important neutrophil gene, Cebpa, during macrophage-neutrophil differentiation. Reporter genes were integrated in a site-specific manner in PUER cells, which are progenitors that can be differentiated into neutrophils or macrophages in vitro by activating the pan-leukocyte TF PU.1. Time series data show that two enhancers upregulate reporter expression during the first 48 hours of neutrophil differentiation. Surprisingly, there is little or no increase in the total accessibility, measured by ATAC-Seq, of the enhancers during the same time period. Conversely, total accessibility peaks 96 hrs after PU.1 activation-consistent with its role as a pioneer-but the enhancers do not upregulate gene expression. Combining deeply sequenced ATAC-Seq data with a new bias-correction method allowed the profiling of accessibility at single-nucleotide resolution and revealed protected regions in the enhancers that match all previously characterized TF binding sites and ChIP-Seq data. Although the accessibility of most positions does not change during early differentiation, that of positions neighboring TF binding sites, an indicator of TF occupancy, did increase significantly. The localized accessibility changes are limited to nucleotides neighboring C/EBP-family TF binding sites, showing that the upregulation of enhancer activity during early differentiation is driven by C/EBP-family TF binding. These results show that increasing the total accessibility of enhancers is not sufficient for upregulating their activity and other events such as TF binding are necessary for upregulation. Also, TF binding can cause upregulation without a perceptible increase in total accessibility. Finally, this study demonstrates the feasibility of comprehensively mapping individual TF binding sites as footprints using high coverage ATAC-Seq and inferring the sequence of events in gene regulation by combining with time-series gene expression data.

MRI-based cell tracking of OATP-expressing cell transplants by pre-labeling with Gd-EOB-DTPA.

Purpose: A critical step in cell-based therapies is determining the exact position of transplanted cells immediately post-transplant. Here, we devised a method to detect cell transplants immediately post-transplant, using a clinical gadolinium-based contrast agent. These cells were detected as hyperintense signals using a clinically familiar T1-weighted MRI protocol. Procedures: HEK293 cells were stably transduced to express human OATP1B3, a hepatic organic anion transporting polypeptide that transports Gd-EOB-DTPA into cells that express the transporters, the intracellular accumulation of which cells causes signal enhancement on T1-weighted MRI. Cells were pre-labeled prior to injection in media containing Gd-EOB-DTPA for MRI evaluation and indocyanine green for cryofluorescence tomography validation. Labeled cells were injected into chicken hearts, in vitro, after which MRI and cryofluorescence tomography were performed in sequence. Results: OATP1B3-expressing cells had substantially reduced T1 following labeling with Gd-EOB-DTPA in culture. Following their implantation into chicken heart, these cells were robustly identified in T1-weighted MRI, with image-derived injection volumes of cells commensurate with intended injection volumes. Cryofluorescence tomography showed that the areas of signal enhancement in MRI overlapped with areas of indocyanine green signal, indicating that MRI signal enhancement was due to the transplanted cells. Conclusions: OATP1B3-expressing cells can be pre-labeled with Gd-EOB-DTPA prior to injection into tissue, affording the use of clinically familiar T1-weighted MRI to robustly detect cell transplants immediately after transplant. This procedure is easily generalizable and has potential advantages over the use of iron oxide based cell labeling agents and imaging procedures.

Antibacterial properties and in vivo efficacy of a novel nitrofuran, IITR06144, against MDR pathogens.

OBJECTIVES: The emergence of MDR Gram-negative pathogens and increasing prevalence of chronic infections presents an unmet need for the discovery of novel antibacterial agents. The aim of this study was to evaluate the biological properties of a small molecule, IITR06144, identified in a phenotypic screen against the Gram-negative model organism Escherichia coli. METHODS: A small-molecule library of 10956 compounds was screened for growth inhibition against E. coli ATCC 25922 at concentration 50 µM. MICs of lead compounds were determined by the broth microdilution method. Time-kill kinetics, anti-persister activity, spontaneous frequency of resistance, biofilm inhibition and disruption were assessed by standard protocols. Resistant mutants were generated by serial passaging followed by WGS. In vitro toxicity studies were carried out via the MTT assay. In vivo toxicity and efficacy in a mouse model were also evaluated. RESULTS: IITR06144 was identified as the most promising candidate amongst 29 other potential antibacterial leads, exhibiting the lowest MIC, 0.5 mg/L. IITR06144 belongs to the nitrofuran class and exhibited broad-spectrum bactericidal activity against most MDR bacteria, including the 'priority pathogen', carbapenem-resistant Acinetobacter baumannii. IITR06144 retained its potency against nitrofurantoin-resistant clinical isolates. It displayed anti-persister, anti-biofilm activity and lack of spontaneous resistance development. IITR06144 demonstrated a large therapeutic index with no associated in vitro and in vivo toxicity. CONCLUSIONS: In the light of excellent in vitro properties displayed by IITR06144 coupled with its considerable in vivo efficacy, further evaluation of IITR06144 as a therapeutic lead against antibiotic-resistant infections is warranted.

The regulatory control of Cebpa enhancers and silencers in the myeloid and red-blood cell lineages.

Cebpa encodes a transcription factor (TF) that plays an instructive role in the development of multiple myeloid lineages. The expression of Cebpa itself is finely modulated, as Cebpa is expressed at high and intermediate levels in neutrophils and macrophages respectively and downregulated in non-myeloid lineages. The cis-regulatory logic underlying the lineage-specific modulation of Cebpa's expression level is yet to be fully characterized. Previously, we had identified 6 new cis-regulatory modules (CRMs) in a 78kb region surrounding Cebpa. We had also inferred the TFs that regulate each CRM by fitting a sequence-based thermodynamic model to a comprehensive reporter activity dataset. Here, we report the cis-regulatory logic of Cebpa CRMs at the resolution of individual binding sites. We tested the binding sites and functional roles of inferred TFs by designing and constructing mutated CRMs and comparing theoretical predictions of their activity against empirical measurements in a myeloid cell line. The enhancers were confirmed to be activated by combinations of PU.1, C/EBP family TFs, Egr1, and Gfi1 as predicted by the model. We show that silencers repress the activity of the proximal promoter in a dominant manner in G1ME cells, which are derived from the red-blood cell lineage. Dominant repression in G1ME cells can be traced to binding sites for GATA and Myb, a motif shared by all of the silencers. Finally, we demonstrate that GATA and Myb act redundantly to silence the proximal promoter. These results indicate that dominant repression is a novel mechanism for resolving hematopoietic lineages. Furthermore, Cebpa has a fail-safe cis-regulatory architecture, featuring several functionally similar CRMs, each of which contains redundant binding sites for multiple TFs. Lastly, by experimentally demonstrating the predictive ability of our sequence-based thermodynamic model, this work highlights the utility of this computational approach for understanding mammalian gene regulation.

The small molecule IITR08027 restores the antibacterial activity of fluoroquinolones against multidrug-resistant Acinetobacter baumannii by efflux inhibition.

Efflux pumps are one of the major determinants of multiple drug resistance. In this study, AbeM, a multidrug efflux pump in Acinetobacter baumannii, was cloned into a multicopy plasmid (pUC18) and was transformed into an efflux-deficient mutant of Escherichia coli (KAM32). The elevated resistance profile of the recombinant E. coli for ciprofloxacin was utilised to screen a small-molecule library of 8000 molecules to identify IITR08027, a small molecule that is not inhibitory on its own but that could potentiate the activity of ciprofloxacin. When used in combination against A. baumannii, the molecule improves the killing efficiency of ciprofloxacin, extends its post-antibiotic effect and causes a decrease in frequency of resistant mutant selection with the antibiotic. Quinacrine-based fluorescence quenching and membrane depolarisation assays revealed that IITR08027 functions as a proton gradient inhibitor. This extends the activity of IITR08027 against other H+-driven efflux pumps such as AbeS. MTT assay against HeLa and HEK293 cells suggested that the molecule is non-toxic at its minimum effective concentration. We propose that, in combination with fluoroquinolones (ciprofloxacin and norfloxacin), IITR08027 may be effective against multidrug-resistant A. baumannii expressing AbeM or other efflux pumps that use the proton gradient as an efflux mechanism.

Fosfomycin resistance in Acinetobacter baumannii is mediated by efflux through a major facilitator superfamily (MFS) transporter-AbaF.

OBJECTIVES: To decipher the function of A1S_1331, named AbaF (Acinetobacter baumannii Fosfomycin efflux), one of the primary targets of AbsR25, a small RNA of A. baumannii. METHODS: abaF was cloned in a multicopy plasmid and expressed from its native promoter in an efflux-deficient strain-Escherichia coli KAM32. Drug susceptibility, accumulation and efflux of ethidium bromide (EtBr) were determined in this strain. abaF was disrupted in A. baumannii using homologous recombination and its effect on drug susceptibility, biofilm formation and virulence was studied. Expression of abaF was followed by quantitative PCR in fosfomycin-challenged A. baumannii and fosfomycin-resistant mutants of A. baumannii. Expression of abaF in clinical strains of A. baumannii was determined by RT-PCR. RESULTS: Expression of abaF in E. coli KAM32 resulted in increased resistance to fosfomycin. Lower accumulation and higher efflux of EtBr from this strain confirmed the role of AbaF as an efflux pump. Disruption of abaF in A. baumannii caused an increase in fosfomycin susceptibility and a decrease in biofilm formation and virulence. The expression of abaF was higher in A. baumannii cells exposed to fosfomycin and in cells resistant to higher concentrations of fosfomycin. The clinically relevant strains of A. baumannii also tested positive for the expression of abaF. CONCLUSIONS: The results of this study suggest that efflux is an important mechanism of fosfomycin resistance and AbaF is involved in fosfomycin resistance in A. baumannii. AbaF also seems to play a role in biofilm formation and virulence of A. baumannii.

Antibacterial potential of a small peptide from Bacillus sp. RPT-0001 and its capping for green synthesis of silver nanoparticles.

Infirmity and death from diseases caused by unsafe food are a continual hazard to communal health safety and socio-economic growth throughout the world. Chemical preservatives are associated with health hazards and toxicity issues. In the study reported here, 200 soil isolates from Western Himalayan region in India were screened for potential antibacterial activity against food-borne pathogens. This study led to the isolation of a bacterial strain belonging to the Genus Bacillus and was designated as RPT-0001. The associated antibacterial activity was sensitive to pronase E treatment. Bioassay-guided fractionation using reverse phase high performance liquid chromatography (RP-HPLC) led to isolation of the antibacterial peptide designated as RPT-0001. The molecular weight of RPT-0001 was determined by electro-spray ionization mass spectroscopy (ESI-MS) as 276.9 Da. RPT-0001 was inhibitory to both Gram-negative and Grampositive food-borne bacteria tested. The characteristics of RPT-0001 do not match with that of any other known antibacterial peptides produced by Bacillus sp. or related genera. Purified RPT-0001 was successfully used in synthesis of silver nanoparticles effective against food-borne pathogenic bacteria. The antibacterial peptide and silver nanoparticles synthesized utilizing it as a capping and reducing agent hold promising potential in food preservation, in packaging material and as a therapeutic agent in the treatment of foodborne infections.

Does a single loading dose of tranexamic acid reduce perioperative blood loss and transfusion requirements after total knee replacement surgery? A randomized, controlled trial.

BACKGROUND: Total knee replacement (TKR) is associated with high-perioperative blood loss, which often requires allogenic blood transfusion. Among the many strategies to decrease the need for allogenic transfusion, tranexamic acid (TA) is used systemically in perioperative setting with promising outcome. Here we evaluated the efficacy of single preoperative bolus dose of TA on reduction in blood loss and red blood cell transfusion in patients undergoing unilateral TKR. MATERIALS AND METHODS: 70, American Society of Anesthesiologists I-II patients scheduled for unilateral TKR were included. Patients were randomly allocated into two groups to receive either TA (Group-TA; 20 mg/kg diluted to 25 cc with normal saline) or an equivalent volume of normal saline (Group P). Hemoglobin concentration, packed cell volume, platelet count, fibrinogen level, D-dimer level was measured preoperatively and at 6(th) and 24(th) h postoperative period. RESULTS: In Group P more blood, colloid and crystalloid solutions were used to replace the blood loss. 27 patients in Group TA did not require transfusion of any blood products compared to 6 patients in Group P (P < 0.0001) and only 3 units of blood was transfused in Group TA where as a total of 32 units of blood was transfused in Group P. Despite the more numerous transfusions, Hb% after 6 h and 24 h in Group P were considerably low in comparison with Group TA (P < 0.0001). CONCLUSION: Tranexamic acid while significantly reducing blood loss caused by TKR surgery collaterally reduced the need for postoperative blood transfusion.

Prevention of altered hemodynamics after spinal anesthesia: A comparison of volume preloading with tetrastarch, succinylated gelatin and ringer lactate solution for the patients undergoing lower segment caesarean section.

BACKGROUND: Spinal anesthesia has replaced general anesthesia in obstetric practice. Hemodynamic instability is a common, but preventable complication of spinal anesthesia. Preloading the circulation with intravenous fluids is considered a safe and effective method of preventing hypotension following spinal anesthesia. We had conducted a study to compare the hemodynamic stability after volume preloading with either Ringer's lactate (RL) or tetrastarch hydroxyethyl starch (HES) or succinylated gelatin (SG) in the patients undergoing cesarean section under spinal anesthesia. MATERIALS AND METHODS: It was a prospective, double-blinded and randomized controlled study. Ninety six ASA-I healthy, nonlaboring parturients were randomly divided in 3 groups HES, SG, RL (n = 32 each) and received 10 ml/kg HES 130/0.4; 10 ml/kg SG (4% modified fluid gelatin) and 20 ml/kg RL respectively prior to SA scheduled for cesarean section. Heart rate, blood pressure (BP), oxygen saturation was measured. RESULTS: The fall in systolic blood pressure (SBP) (<100 mm Hg) noted among 5 (15.63%), 12 (37.5%) and 14 (43.75%) parturients in groups HES, SG, RL respectively. Vasopressor (phenylephrine) was used to treat hypotension when SBP <90 mm Hg. Both the results and APGAR scores were comparable in all the groups. Lower preloading volume and less intra-operative vasopressor requirement was noted in HES group for maintaining BP though it has no clinical significance. CONCLUSION: RL which is cheap, physiological and widely available crystalloid can preload effectively and maintain hemodynamic stability well in cesarean section and any remnant hypotension can easily be manageable with vasopressor.

The efficacy of ginger added to ondansetron for preventing postoperative nausea and vomiting in ambulatory surgery.

BACKGROUND: Post-operative nausea and vomiting (PONV) frequently hampers implementation of ambulatory surgery in spite of so many costly antiemetic drugs and regimens. OBJECTIVE: The study was carried out to compare the efficacy of ginger (Zingiber officinale) added to Ondansetron in preventing PONV after ambulatory surgery. MATERIALS AND METHODS: It was a prospective, double blinded, and randomized controlled study. From March 2008 to July 2010, 100 adult patients of either sex, aged 20-45, of ASA physical status I and II, scheduled for day care surgery, were randomly allocated into Group A[(n = 50) receiving (IV) Ondansetron (4 mg) and two capsules of placebo] and Group B[(n = 50) receiving IV Ondansetron (4 mg) and two capsules of ginger] simultaneously one hour prior to induction of general anaesthesia (GA) in a double-blind manner. One ginger capsule contains 0.5 gm of ginger powder. Episodes of PONV were noted at 0.5h, 1h, 2h, 4h, 6h, 12h and 18h post- operatively. STATISTICAL ANALYSIS AND RESULTS: Statistically significant difference between groups A and B (P < 0.05), was found showing that ginger ondansetron combination was superior to plain Ondansetron as antiemetic regimen for both regarding frequency and severity. CONCLUSION: Prophylactic administration of ginger and ondansetron significantly reduced the incidence of postoperative nausea and vomiting compared to ondansetron alone in patients undergoing day care surgery under general anaesthesia.

Evaluating ranitidine, pantoprazole and placebo on gastric pH in elective surgery.

BACKGROUND AND AIM: Concern about the grim nature of postoperative acid aspiration syndrome grew among the anesthesiologist over the years warranting the need for pre-emptive intervention. The aim of the study is to compare the effects of preoperative oral ranitidine versus pantoprazole given in regulating gastric pH in elective surgery. METHODS: This prospective, parallel group, controlled, randomized, single-blind study was conducted at a tertiary care postgraduate teaching institute at Kolkata, involving 120 participants of either sex, aged 18-60 years of American Society of Anesthesiologists physical status I and II, who were scheduled for elective surgery under general anesthesia lasting for more than 2 h. The participants were divided into three groups. In group A (n=40) participants received placebo tablet, in group B (n=40) participants received ranitidine tablet while in group C (n=40), participants received pantoprazole tablet and their gastric pH estimated serially. RESULTS: The participants in the three groups were comparable in terms of age, sex, body weight, duration of surgery and type of surgery distribution. In regard to changes in gastric pH trends, there was no statistically significant difference between serial pH values in group A (Friedman test; P>0.05) and group C participants. (P>0.05). However, the mean preoperative gastric pH values (7.140±.7652) were significantly lower than mean pH values (7.253±.7514) after 2 h postoperatively in group B participants (P<0.05). CONCLUSION: From the observations and analyses of the present study, it can be inferred that ranitidine is more effective than pantoprazole to raise the gastric pH for prevention of aspiration pneumonitis.

GIS based water quality indexing of Malad creek, Mumbai (India): an impact of sewage discharges.

Malad creek is one of the most heavily polluted water bodies in Mumbai, India. Presently, creek receives wastewater and sewage from open drains and nallahs as well as partially treated wastewater from treatment facilities. The objective of the present study was to assess and classify the water quality zones spatially and temporally based on physico-chemical and bacteriological analysis. For this, GIS based methodology was integrated with water quality indexing, according to National Sanitation Foundation. Nine water quality parameters were considered to generate the indices that represent the overall status of creek water quality. Based on field observations and spatial distribution of water quality, various options were suggested for improvement in water quality of the creek.