Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets▿

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria

Turismo de vida silvestre: una síntesis de la agenda de investigación pasada, presente y futuro

Wildlife tourism (WT) is an emerging sector of tourism, majorly meant to view and/or encounter wildlife in the wild, captive, and semi-captive settings. Because of the new emerging economies, there is an increased demand for wildlife destinations in both, developing and developed nations. However, a comprehensive study is lacking in WT. In this context, the present study seeks to bring together and discuss the key findings on WT from the present literature and propose new approaches to research using co-citation, co-authorship, and co-occurrence analyses. Further, the study also considers research on WT conducted so far like attitudes, bird-watching, conservation, economics, hunting, mammals, management, marine monitoring, negative impacts, positive impacts, captive wildlife, and guidelines. A data set is created that includes authors, article titles, citations, countries, co-authorship, institutions, publication years and sources, keywords, and abstracts by collecting the bibliographies from Scopus and Web of Science (WoS) indexed journals with keywords search “Wild Life, Jungle and Tourism.” The study collected 1,519 and used 1,259 published articles from 1990 to 2020, and analyzed employing VOS viewer software, which has enabled us to understand the relationship and structure of the literatureEl turismo de vida silvestre (WT) es un sector emergente del turismo, destinado principalmente a ver y / o encontrar vida silvestre en entornos silvestres, cautivos y semi-cautivos. Debido a las nuevas economías emergentes, existe una mayor demanda de destinos de vida silvestre tanto en países desarrollados como en desarrollo. Sin embargo, falta un estudio completo en WT. En este contexto, el presente estudio busca reunir y discutir los hallazgos clave sobre WT de la literatura actual y proponer nuevos enfoques de investigación utilizando análisis de co-cita, coautoría y co-ocurrencia. Además, el estudio también considera la investigación sobre WT realizada hasta ahora como actitudes, observación de aves, conservación, economía, caza, mamíferos, manejo, monitoreo marino, impactos negativos, impactos positivos, vida silvestre cautiva y pautas. Se crea un conjunto de datos que incluye a los autores, títulos de artículos, citas, países, coautoría, instituciones, años de publicación y fuentes, palabras clave y resúmenes mediante la recopilación de bibliografías de revistas indexadas de Scopus y Web of Science (WoS) con la búsqueda de palabras clave "Wild Life, Jungle and Tourism". El estudio recopiló 1.519 y utilizó 1.259 artículos publicados entre 1990 y 2020, y analizó utilizando el software de visualización VOS, lo que nos ha permitido comprender la relación y la estructura de la literatur

Development of micro-shock wave assisted dry particle and fluid jet delivery system

Small quantity of energetic material coated on the inner wall of a polymer tube is proposed as a new method to generate micro-shock waves in the laboratory. These micro-shock waves have been harnessed to develop a novel method of delivering dry particle and liquid jet into the target. We have generated micro-shock waves with the help of reactive explosive compound high melting explosive (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine) and traces of aluminium] coated polymer tube, utilising 9 J of energy. The detonation process is initiated electrically from one end of the tube, while the micro-shock wave followed by the products of detonation escape from the open end of the polymer tube. The energy available at the open end of the polymer tube is used to accelerate tungsten micro-particles coated on the other side of the diaphragm or force a liquid jet out of a small cavity filled with the liquid. The micro-particles deposited on a thin metal diaphragm (typically 100-mu m thick) were accelerated to high velocity using micro-shock waves to penetrate the target. Tungsten particles of 0.7 mu m diameter have been successfully delivered into agarose gel targets of various strengths (0.6-1.0 %). The device has been tested by delivering micro-particles into potato tuber and Arachis hypogaea Linnaeus (ground nut) stem tissue. Along similar lines, liquid jets of diameter 200-250 mu m (methylene blue, water and oils) have been successfully delivered into agarose gel targets of various strengths. Successful vaccination against murine salmonellosis was demonstrated as a biological application of this device. The penetration depths achieved in the experimental targets are very encouraging to develop a future device for biological and biomedical applications

Acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella

During the course of infection, Salmonella has to face several potentially lethal environmental conditions, one such being acidic pH. The ability to sense and respond to the acidic pH is crucial for the survival and replication of Salmonella. The physiological role of one gene (STM1485) involved in this response, which is upregulated inside the host cells (by 90- to 113-fold) is functionally characterized in Salmonella pathogenesis. In vitro, the DSTM1485 neither exhibited any growth defect at pH 4.5 nor any difference in the acid tolerance response. The DSTM1485 was compromised in its capacity to proliferate inside the host cells and complementation with STM1485 gene restored its virulence. We further demonstrate that the surface translocation of Salmonella pathogenicity island-2 (SPI-2) encoded translocon proteins, SseB and SseD were reduced in the DSTM1485. The increase in co-localization of this mutant with lysosomes was also observed. In addition, the DSTM1485 displayed significantly reduced competitive indices (CI) in spleen, liver and mesenteric lymph nodes in murine typhoid model when infected by intra-gastric route. Based on these results, we conclude that the acidic pH induced STM1485 gene is essential for intracellular replication of Salmonella

Antimony-resistant but not antimony-sensitive Leishmania donovani up-regulates host IL-10 to overexpress multidrug-resistant protein 1

The molecular mechanism of antimony-resistant Leishmania donovani (SbRLD)–driven up-regulation of IL-10 and multidrug-resistant protein 1 (MDR1) in infected macrophages (Mϕs) has been investigated. This study showed that both promastigote and amastigote forms of SbRLD, but not the antimony-sensitive form of LD,express a unique glycan with N-acetylgalactosamine as a terminal sugar. Removal of it either by enzyme treatment or by knocking down the relevant enzyme, galactosyltransferase in SbRLD (KDSbRLD), compromises the ability to induce the above effects. Infection of Mϕs with KD SbRLD enhanced the sensitivity towardantimonials compared with infection with SbRLD, and infection of BALB/c mice with KD SbRLD caused significantly less organ parasite burden compared with infection induced by SbRLD. The innate immune receptor, Toll-like receptor 2/6 heterodimer, is exploited by SbRLD to activate ERK and nuclear translocation of NF-κB involving p50/c-Rel leading to IL-10 induction, whereas MDR1 up-regulation is mediated by PI3K/Akt and the JNK pathway.Interestingly both recombinant IL-10 and SbRLD up-regulate MDR1 in Mϕ with different time kinetics, where phosphorylation of PI3K was noted at 12 h and 48 h, respectively, but Mϕs derived from IL-10−/− mice are unable to show MDR1 up-regulation on infection with SbRLD. Thus, it is very likely that an IL-10 surge is a prerequisite for MDR1 up-regulation. The transcription factor important for IL-10–driven MDR1 up-regulation is c-Fos/c-Jun and not NF-κB, as evident from studies with pharmacological inhibitors and promoter mapping with deletion constructs

Needleless Vaccine Delivery Using Micro-Shock Waves ▿ †

Shock waves are one of the most efficient mechanisms of energy dissipation observed in nature. In this study, utilizing the instantaneous mechanical impulse generated behind a micro-shock wave during a controlled explosion, a novel nonintrusive needleless vaccine delivery system has been developed. It is well-known that antigens in the epidermis are efficiently presented by resident Langerhans cells, eliciting the requisite immune response, making them a good target for vaccine delivery. Unfortunately, needle-free devices for epidermal delivery have inherent problems from the perspective of the safety and comfort of the patient. The penetration depth of less than 100 μm in the skin can elicit higher immune response without any pain. Here we show the efficient utilization of our needleless device (that uses micro-shock waves) for vaccination. The production of liquid jet was confirmed by high-speed microscopy, and the penetration in acrylamide gel and mouse skin was observed by confocal microscopy. Salmonella enterica serovar Typhimurium vaccine strain pmrG-HM-D (DV-STM-07) was delivered using our device in the murine salmonellosis model, and the effectiveness of the delivery system for vaccination was compared with other routes of vaccination. Vaccination using our device elicits better protection and an IgG response even at a lower vaccine dose (10-fold less) compared to other routes of vaccination. We anticipate that our novel method can be utilized for effective, cheap, and safe vaccination in the near future

Tristetraprolin Mediates Radiation-Induced TNF-α Production in Lung Macrophages

<div><p>The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For <i>in vitro</i> studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the <i>in vivo</i> relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from <i>ttp</i> (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTP^Ser178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.</p> </div

Radiation-induced TTP degradation and TNF-α secretion is inhibited significantly by the proteasome inhibitor MG132.

<p>(A) MH-S cells were metabolically labeled with ³⁵S-methionine, were either sham-irradiated or irradiated with 4 Gy, and chased with cold methionine for indicated time periods. After the chase period cell lysates were subjected to immunoprecipitation using TTP antibody, immunocomplexes were resolved by SDS-PAGE and autoradiography. (B) TTP protein’s half life in sham-irradiated (-RT) and irradiated (4 Gy) groups were determined by densitometric scanning of the autoradiographs followed by quantitation using Image J1.32j software (NIH, Bethesda, MD). Relative protein levels were determined in comparison to sample isolated immediately after the pulse labeling (0 h chase). (C) MH-S cells were either sham-irradiated or radiated with 4 Gy, and 44 h after radiation 2 µM of MG132 was added. Cell lysates were collected 4 h after MG132 addition and immunoblotted for TTP and TNF-α. GAPDH was used as loading control.</p

p38 kinase controls radiation-induced TTP phosphorylation and TNF-α secretion by MH-S cells.

<p>(A) CHO cells overexpressing TTP were treated with 4 Gy in the presence of either DMSO (vehicle control) or p38 inhibitor (SB203580), or PI3K inhibitor (Wortmannin), or GSK3ß inhibitors (SB415286, SB216763). Cell lysates were prepared 10 min post-radiation and immunoblotted using indicated antibodies. (B) MH-S cells were pretreated with either p38 or GSK3ß inhibitors as above and cell lysates were prepared 10 min post-radiation and immunoblotted with the indicated antibodies. (C) MH-S cells were irradiated with 4 Gy in the presence or absence of a p38 inhibitor (SB203580), and radiation-induced TNF-α secretion was quantified using ELISA. (D) MH-S cells were treated with either control (C) or TTP (T) siRNA. 24 h post-transfection, cells were either left un-irradiated or radiated with 4 Gy. Culture supernatants were collected 48 h post-radiation, and TNF-α levels were quantified. In the inset, the effectiveness of p38 siRNA is shown in cell lysates isolated from C or T siRNA treated cells.</p

Schematic model explaining the role of post-translational TTP modifications (phosphorylation and degradation) in radiation-induced TTP inactivation as an upstream regulator involved in increased TNF-α secretion by mouse lung macrophages.

<p>Schematic model explaining the role of post-translational TTP modifications (phosphorylation and degradation) in radiation-induced TTP inactivation as an upstream regulator involved in increased TNF-α secretion by mouse lung macrophages.</p