Identification and Characterization of the Insecticidal Toxin “Makes Caterpillars Floppy” in Photorhabdus temperata M1021 Using a Cosmid Library

Photorhabdus temperata is an entomopathogenic enterobacterium; it is a nematode symbiont that possesses pathogenicity islands involved in insect virulence. Herein, we constructed a P. temperata M1021 cosmid library in Escherichia coli XL1-Blue MRF` and obtained 7.14 × 10⁵ clones. However, only 1020 physiologically active clones were screened for insect virulence factors by injection of each E. coli cosmid clone into Galleria mellonella and Tenebrio molitor larvae. A single cosmid clone, PtC1015, was consequently selected due to its characteristic virulent properties, e.g., loss of body turgor followed by death of larvae when the clone was injected into the hemocoel. The sequence alignment against the available sequences in Swiss-Prot and NCBI databases, confirmed the presence of the mcf gene homolog in the genome of P. temperata M1021 showing 85% homology and 98% query coverage with the P. luminescens counterpart. Furthermore, a 2932 amino acid long Mcf protein revealed limited similarity with three protein domains. The N-terminus of the Mcf encompassed consensus sequence for a BH3 domain, the central region revealed similarity to toxin B, and the C-terminus of Mcf revealed similarity to the bacterial export domain of ApxIVA, an RTX-like toxin. In short, the Mcf toxin is likely to play a role in the elimination of insect pests, making it a promising model for use in the agricultural field.     

Leishmania-induced biphasic ceramide generation in macrophages is crucial for uptake and survival of the parasite

The initial macrophage-Leishmania donovani interaction results in the formation of membrane platforms, termed lipid rafts, that help in the entry of the parasite. Therefore, it is imperative that the parasite designs a strategy to modulate its uptake and survival within the macrophages. Herein, we report Leishmania-triggered biphasic ceramide generation. In the first phase, L. donovani promastigotes induce activation of acid sphingomyelinase (ASMase), which catalyzes the formation of ceramide from sphingomyelin. Inhibition of ASMase resulted in reduced uptake and infection with the parasite. In the second phase, de novo synthesis generates ceramide that reduces the cellular cholesterol level and displaces the cholesterol from the membrane, leading to enhanced membrane fluidity, disruption of rafts, and impaired antigen-presentation to the T cells. The results reveal a novel role for ceramide in the perspective of L. donovani infection and help formulate an antileishmanial strategy that can possibly be applied to other intracellular infections as well.     

Efficacy and safety of,and patient satisfaction with,colonoscopic-administered fecal microbiota transplantation in relapsing and refractory community- and hospital-acquired Clostridium difficile infection

OBJECTIVE:

To report the efficacy and safety of, and patient satisfaction with, colonoscopic fecal microbiota transplantation (FMT) for community- and hospital-acquired Clostridium difficile infection (CDI).

METHODS:

A retrospective medical records review of patients who underwent FMT between July 1, 2012 and August 31, 2013 was conducted. A total of 22 FMTs were performed on 20 patients via colonoscopy. The patients were divided into ‘community-acquired’ and ‘hospital-acquired’ CDI. Telephone surveys were conducted to determine procedure outcome and patient satisfaction. Primary cure rate was defined as resolution of diarrhea without recurrence within three months of FMT, whereas secondary cure rate described patients who experienced resolution of diarrhea and return of normal bowel function after a second course of FMT.

RESULTS:

Nine patients met the criteria for community-acquired CDI whereas 11 were categorized as hospital-acquired CDI. A female predominance in the community-acquired group (88.89% [eight of nine]) was found (P=0.048). The primary cure rate was 100% (nine of nine) and 81.8% (nine of 11 patients) in community- and hospital-acquired CDI groups, respectively (P=0.189). Two patients in the hospital-acquired group had to undergo a repeat FMT for persistent symptomatic infection; the secondary cure rate was 100%. During the six-month follow-up, all patients were extremely satisfied with the procedure and no complications or adverse events were reported.

CONCLUSION:

FMT was a highly successful and very acceptable treatment modality for treating both community- and hospital-acquired CDI.     

Phytochemical analysis and radical scavenging profile of juices of <Emphasis Type="Italic">Citrus sinensis,Citrus anrantifolia</Emphasis>, and <Emphasis Type="Italic">Citrus limonum</Emphasis>

Background

The aim of the current investigation was to identify bioactive secondary metabolites including phenols, tannins, flavonoids, terpinedes, and steroids and compare the phytochemical analysis and antioxidant profile of the juice extracted from the fruits of Citrus sinensis, Citrus anrantifolia, and Citrus limonum.

Results

Phytochemical screening is important for the isolation of new, novel, and rare secondary metabolites before bulk extraction. Phytochemical analysis of the desired plant fruits of family Rutaceae revealed the presence of phenols, flavonoids, reducing sugars, steroids, terpinedes and tannins. The fruits of C. sinensis and C. anrantifolia exhibited the presence of phenols, flavonoids, reducing sugars, steroids, terpinedes and tannins, while the fruits of C. limonum indicated the presence of phenols, flavonoids, reducing sugars, terpinedes, and tannins. The fruits of selected plants were also subjected to antioxidant potential by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay against ascorbic acid at various concentrations. Among the tested plants, C. sinensis showed promising antiradical effect (84.81%) which was followed by C. Anrantifolia (80.05%) at 100 μg/ml against ascorbic acid (96.36%). The C. limonum showed low antioxidant activity among the three selected plants of family Rutaceae.

Conclusions

The current finding is baseline information in the use of the fruits of selected plants as food supplement which may be due to the presence of antioxidant molecules in the family Rutaceae. Further research is needed in this area to isolate the phenolic constituents which possess ideal antiradical potential.

     

Inhibition of Autoregulated TGFβ Signaling Simultaneously Enhances Proliferation and Differentiation of Kidney Epithelium and Promotes Repair Following Renal Ischemia

We studied autocrine transforming growth factor (TGF)β signaling in kidney epithelium. Cultured proximal tubule cells showed regulated signaling that was high during log-phase growth, low during contact-inhibited differentiation, and rapidly increased during regeneration of wounded epithelium. Autoregulation of signaling correlated with TGFβ receptor and Smad7 levels, but not with active TGFβ, which was barely measurable in the growth medium. Confluent differentiated cells with low receptor and high Smad7 levels exhibited blunted responses to saturating concentrations of exogenously provided active TGFβ, suggesting that TGFβ signaling homeostasis was achieved by cell density-dependent modulation of signaling intermediates. Antagonism of Alk5 kinase, the TGFβ type I receptor, dramatically accelerated the induction of differentiation in sparse, proliferating cultures and permitted better retention of differentiated features in regenerating cells of wounded, confluent cultures. Alk5 antagonism accelerated the differentiation of cells in proximal tubule primary cultures while simultaneously increasing their proliferation. Consequently, Alk5-inhibited primary cultures formed confluent, differentiated monolayers faster than untreated cultures. Furthermore, treatment with an Alk5 antagonist promoted kidney repair reflected by increased tubule differentiation and decreased tubulo-interstitial pathology during the recovery phase following ischemic injury in vivo. Our results show that autocrine TGFβ signaling in proliferating proximal tubule cells exceeds the levels that are necessary for physiological regeneration. To that end, TGFβ signaling is redundant and maladaptive during tubule repair by epithelial regeneration.Regeneration of an adult epithelium such as those lining the kidney tubules involves not only proliferation but also de-differentiation, followed by growth arrest and re-differentiation.^1,2,3 The signaling cues that coordinate these processes are largely unknown. Endocrine and paracrine factors affect epithelial repair following injury in vivo. Nevertheless, epithelial homeostasis is also regulated by density-dependent contact-inhibition and attendant differentiation. The mechanisms that mediate these processes are poorly understood, but likely involve transforming growth factor (TGF)β, in addition to signals generated by the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. These considerations prompted us to investigate how endogenously generated signals might control epithelial regeneration in terms of cell proliferation and differentiation.Increased TGFβ signaling can lead to apoptosis, growth inhibition, or epithelial-mesenchymal transitions (EMT) of epithelial cells, including kidney epithelial cells.^4,5,6,7,8,9 Prolonged exposure to high concentrations of active TGFβ is frequently used to model these alterations. While these effects of sustained high intensity TGFβ signaling are well studied, much less is known about physiologically regulated TGFβ signals and how they become increased by epithelial injury and subsequent regeneration. Autocrine TGFβ signals are antiproliferative for epithelial cells and cultures from TGFβ₁ null kidney tubules display enhanced proliferative rates.¹⁰ Nevertheless, TGFβ signaling was found to be increased rather than decreased during the proliferation of surviving kidney epithelium following cell loss by ischemia, and this was accompanied by increased expression of TGFβ and its receptors in regenerating cells.¹¹ Similarly, proliferating keratinocytes in skin wounds show enhanced TGFβ signaling¹². It has been puzzling why antiproliferative TGFβ signaling becomes enhanced in rapidly growing cells under pathological conditions.In this study, we have investigated the functional relevance of cell-autonomous, ie, endogenously generated, TGFβ signals for regenerating kidney epithelial cells in culture and in vivo. Fully differentiated proximal tubule (PT) cells retain the ability to undergo mitotic division,^13,14,15 and, following cell loss by injury, survivors dedifferentiate, proliferate, and then redifferentiate to reconstitute the lost cell mass.^1,2,3,13,16 We found that cell-autonomous TGFβ signals are tightly autoregulated during repeated cycles of proliferation and contact-inhibition in PT cultures. Signaling was high during log phase growth and became progressively suppressed as cultures became contact-inhibited and differentiated. It was decreased in growing subconfluent cultures by neutralizing TGFβ antibodies, indicating a requirement for extracellular ligand. However, in the absence of TGFβ antibodies, increases and decreases of signaling were determined solely by cell density, and occurred independently of the concentrations of barely measurable active TGFβ in growth medium. Instead, the signaling fluctuations were associated with increased and decreased expression of TGFβ receptor and reciprocal alterations of inhibitory Smad7. Moreover, saturating concentrations of exogenous TGFβ were found to elicit blunted signaling responses from contact-inhibited differentiated cells relative to growing undifferentiated cells. These observations suggested that: (1) extracellular TGFβ ligand played a permissive role but did not, by itself, determine the intensity of signaling fluctuations during the epithelial growth cycle; and (2) signaling homeostasis during growth and quiescence was related to the modulation of TGFβ receptors and Smad7. Functionally, we found that inhibition of cell-autonomous TGFβ signals resulted in remarkably accelerated differentiation and concurrent stimulation of proliferation in growing PT cultures. Importantly, we extended our observations to demonstrate that treatment with small molecule Alk5 inhibitors not only promoted differentiation in regenerating PT epithelium during wound healing in vitro, but also improved the repair of kidney damage with greater restoration of epithelial differentiation and tubule integrity following ischemia in vivo. These unprecedented findings have direct relevance to the development of treatments that might promote repair and recovery following loss of epithelium by acute kidney injury (AKI).