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Randia aculeata (Rubiaceae) is a medicinal plant distributed in the state of Jalisco, Mexico. In folk medicine it is used to counteract the effects produced by the bite of snakes and other poisonous insects. However, there are no reports of new alternatives to use the plant in a sustainable way. The main objective of this study was to establish a cell culture of R. aculeata, evaluate its anti-inflammatory and antibacterial potential and determine the phytochemical profile. In addition, we determine the total phenolics content and antioxidant activity of methanol extracts. Cell suspension cultures (CSC) was established with 2.0 mg/L 1-naphthaleneacetic acid and 0.5 mg/L 6-furfurylaminopurine in MS culture medium. CSC produced 13.77 ± 0.49 g/L biomass in dry weight at 36 days of culture. GC-MS analysis showed high amounts of β-sitosterol for hexane (HE-L: 22.02 ± 0.44%) and dichloromethane (DE-L: 19.69 ± 0.49%) leaf extracts. Hexane CSC extract (HE-CSC) stood out for containing high amount of palmitic acid (43.95 ± 0.02%) and dichloromethane extract (DE-CSC) had mainly palmitic acid (30.16 ± 2.14%) and vanillic acid (10.23 ± 0.34%). The methanol CSC extract (ME-CSC) showed antioxidant activity with an IC50 value of 16.49 ± 0.25 µg/mL, which was almost equal to the methanol leaf extract (ME-L) that showed a IC50 of 14.37 ± 0.20 µg/mL. The HE-CSC and DE-CSC were the only ones that showed inhibition against S. aureus, S. aureus-MRSA, S. typhimurium and E. coli. However, S. aureus was the most susceptible with HE-CSC (MIC = 62.5 µg/mL) and DE-CSC (MIC = 125 µg/mL). The HE-CSC exhibited the highest percentages of inhibition of NO production (36.88 ± 4.35%) at 40 µg/mL in RAW 264.7 macrophages, which was slightly lower than indomethacin (47.45 ± 7.41%) tested at 30 µg/mL. This extract was also the most effective against bacteria. The results suggest that cell culture extracts of R. aculeata may be a sustainable source for obtaining bioactive compounds.

Most bacteria are protected from environmental offenses by a cell wall consisting of strong yet elastic peptidoglycan. The cell wall is essential for preserving bacterial morphology and viability, and thus the enzymes involved in the production and turnover of peptidoglycan have become preferred targets for many of our most successful antibiotics. In the past decades, Vibrio cholerae, the gram-negative pathogen causing the diarrheal disease cholera, has become a major model for understanding cell wall genetics, biochemistry, and physiology. More than 100 articles have shed light on novel cell wall genetic determinants, regulatory links, and adaptive mechanisms. Here we provide the first comprehensive review of V. cholerae’s cell wall biology and genetics. Special emphasis is placed on the similarities and differences with Escherichia coli, the paradigm for understanding cell wall metabolism and chemical structure in gram-negative bacteria.

Graphical abstract, Highlights • Subchronic HCB-administration in male rats generates arterial hypertension. • AT1 activity is involved in HCB- induced hypertension. • AT1 activity is involved in arterial morphological changes caused by HCB. • AT1 activity is involved in HCB- produced alterations in arterial relaxation. • Losartan prevents HCB-produced alterations in TGF-β1, eNOS and AT1 expressions., Environmental hexachlorobenzene (HCB) increases blood pressure (BP) in female rats, causing alterations in arterial structure and function. Here we study the role of Angiotensin II receptor type 1 (AT1) in HCB-induced hypertension through the use of AT1 antagonist losartan. HCB-treated male rats showed a 22.7% increase in BP which was prevented by losartan. Losartan blocked HCB-induced changes in arterial morphology (decreased aorta cell number and increased wall thickness). Losartan also prevented HCB-induced alterations in artery relaxation by acetylcholine and nitroprusside but not the reduction in the maximum contraction by phenylephrine. Losartan rescued arterial molecular alterations caused by HCB (i.e. an increase in TGF-β1 and AT1 expression and a decrease in eNOS expression and nitrite levels) and reduced hydrogen sulfide plasma concentration. In conclusion: in this work we demonstrate that AT1 activity is involved in HCB effects on the vascular system leading to hypertension.

AIMS To determine if the combination of exercise and statin could normalize postprandial triglyceridaemia (PPTG) in hypercholesteraemic individuals. METHODS Eight hypercholesteraemic (blood cholesterol 182 ± 38 mg dL-1 ; low-density lipoprotein-cholesterol [LDL-c] 102 ± 32 mg dL-1 ) overweight (body mass index 30 ± 4 kg m-2 ) individuals with metabolic syndrome (MetS) were compared to a group of 8 metabolically healthy (MetH) controls (blood cholesterol 149 ± 23 mg dL-1 ; LDL-c 77 ± 23 mg dL-1 , and body mass index 23 ± 2 kg m-2 ). Each group underwent 2 PPTG tests, either 14 hours after a bout of intense exercise or without previous exercise. Additionally, MetS individuals were tested 96 hours after withdrawal of their habitual statin medication to study medication effects. RESULTS A bout of exercise before the test meal did not reduce PPTG in MetS (P = .347), but reduced PPTG by 46% in MetH (413 ± 267 to 224 ± 142 mg dL-1 for 5 h incremental area under the curve; P = .02). In both trials (i.e., either after a bout of intense exercise or without previous exercise), statin withdrawal in MetS greatly increased PPTG (average 65%; P < .01), mean LDL-c (average 25%; P < .01), total cholesterol (average 16%; P < .01) and apolipoprotein (Apo) B48 (24%; P < .01), without interference from exercise. However, Apo B100 was not affected by statin withdrawal. CONCLUSION Hypercholesteraemic MetS individuals (compared to MetH controls) fail to show an effect of exercise on reducing PPTG. However, chronic statin medication blunts the elevations in triglyceride after a fat meal (i.e., incremental area under the curve of PPTG) reducing their cardiovascular risk associated with their atherogenic dyslipidaemia. Statin decreases PPTG by reducing the secretion or accelerating the catabolism of intestinal Apo B48.

Cervical cancer is the fourth most frequently occurring malignancy in women worldwide. It is of vital importance to find new alternatives for the treatment of this disease. Isatin is a natural compound with a molecular versatile architecture, which allows its use in a wide range of fields in medicinal chemistry. Many of these compounds have been evaluated as anticancer agents in many cell lines; however, their action mechanism remains uncertain. In this work, we investigated 55 isatin derivatives with reported cytotoxic activity against the human cervical cancer cell line (HeLa), to propose their action mechanism. As a first step, to unravel the key structural features of these compounds that result in anticancer activity over the HeLa line, a QSAR study was done. Our QSAR model revealed that the positive ionizable groups, planar molecular shape and hydrophobic character define the potency of the compounds. The statistical parameters values of our QSAR model were Q2 = 87.5, R2 = 91.24, s = 0.165 and F = 52.08; additionally, the QUIK, redundancy and overfitting rules were applied. The predictive ability of the model was evaluated using 30% of the compounds in this study as the external set, obtaining a Q2ext = 79.99 value. From these results, a molecular similarity analysis (MSA) of the isatin derivatives with a series of DNA intercalating and minor groove-binding agents, and further docking with DNA, suggested that the molecular action mechanism of these compounds may be as DNA minor groove-binder agents in assays with the HeLa cell line.

A synthesis by plasma of polymers with structure of oxidized ethylene (PPEO) obtained from ethanol is presented in this work with the objective to obtain hydrophilic and potentially biodegradable PPEO films. The synthesis was carried with rf glow discharges in the 20–120 W interval obtaining films with a 0.38 μm/W growing rate thickness. Morphology, chemical structure, hydrophilicity and absorption of electromagnetic energy in the UV–visible interval were analyzed. The results indicated that PPEO has smooth surfaces with particles and/or folded areas with water contact angles between 72° and 97°, in the limit between hydrophilicity and hydrophobicity. The structural chemical analysis showed the presence of C–H and O–H bonds characteristic of the monomer, and C=O and ≈C≈ groups characteristics of plasma polymers with dehydrogenation and formation of multiple bonds. The absorption of electromagnetic energy of PPEO was more intense in the UV than in the visible wavelength interval. These characteristics indicate that PPEO can be biodegradable under the sunlight and/or under wet environments.

The peptidoglycan cell wall is a predominant defining structure of bacteria, determining cell shape and supporting survival in diverse conditions. As a single, macromolecular sacculus enveloping the bacterial cell during growth and division, peptidoglycan is necessarily a dynamic structure that requires highly regulated synthesis of new material, remodeling, and turnover, or autolysis, of old material. Despite ubiquitous clinical exploitation of peptidoglycan synthesis as an antibiotic target, much remains unknown about how bacteria modulate synthetic and autolytic processes. Here, we couple bacterial genetics in Vibrio cholerae with compositional analysis of soluble pools of peptidoglycan turnover products to uncover a critical role for a widely misunderstood class of autolytic enzymes, the lytic transglycosylases (LTGs). We demonstrate that LTG activity is specifically required for vegetative growth. The vast majority of LTGs, however, are dispensable for growth, and defects that are ultimately lethal accumulate due to generally inadequate LTG activity, rather than the absence of specific individual enzymes. Consistent with this, we found that a heterologously expressed E. coli LTG, MltE, is capable of sustaining V. cholerae growth in the absence of endogenous LTGs. Lastly, we demonstrate that soluble, uncrosslinked, endopeptidase-dependent peptidoglycan chains accumulate in the WT, and, to a higher degree, in LTG mutants, and that LTG mutants are hyper-susceptible to the production of diverse periplasmic polymers. Collectively, our results suggest that a key function of LTGs is to prevent toxic crowding of the periplasm with synthesis-derived PG fragments. Contrary to prevailing models, our data further suggest that this process can be temporally separate from peptidoglycan synthesis.

Synthesis and turnover of the bacterial cell wall must be tightly coordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines, the aPBPs and the Rod system., The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth, and structural integrity. PG is synthesized by class A/B penicillin-binding proteins (a/bPBPs) and shape, elongation, division, and sporulation (SEDS) proteins like RodA (as part of the Rod system cell elongation machinery) and degraded by “autolytic” enzymes to accommodate growth processes. It is thought that autolysins (particularly endopeptidases [EPs]) are required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and PG synthesis remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological and division defects, but these cells continued to increase in mass and aberrantly incorporated new cell wall material. Mass increase proceeded in the presence of Rod system inhibitors, but cells lysed upon inhibition of aPBPs, suggesting that aPBPs are required for structural integrity under these conditions. The Rod system, although not essential for the observed mass increase, remained functional even after prolonged EP depletion. Last, heterologous expression of an EP from Neisseria gonorrhoeae fully complemented growth and morphology of an EP-insufficient V. cholerae, highlighting the possibility that the PG synthases may not necessarily function via direct interaction with EPs. Overall, our findings suggest that during EP insufficiency in V. cholerae, aPBPs become essential for structural integrity while the Rod system is unable to promote proper cell expansion.

Peptidoglycan (PG) is essential for bacterial survival and maintaining cell shape. The rod-shaped model bacterium Escherichia coli has a set of seven endopeptidases that remodel the PG during cell growth. The gamma proteobacterium Candidatus Thiosymbion oneisti is also rod-shaped and attaches to the cuticle of its nematode host by one pole. It widens and divides by longitudinal fission using the canonical proteins MreB and FtsZ. The PG layer of Ca. T. oneisti has an unusually high peptide cross-linkage of 67% but relatively short glycan chains with an average length of 12 disaccharides. Curiously, it has only two predicted endopeptidases, MepA and PBP4. Cellular localization of symbiont PBP4 by fluorescently labeled antibodies reveals its polar localization and its accumulation at the constriction sites, suggesting that PBP4 is involved in PG biosynthesis during septum formation. Isolated symbiont PBP4 protein shows a different selectivity for β-lactams compared to its homologue from E. coli. Bocillin-FL binding by PBP4 is activated by some β-lactams, suggesting the presence of an allosteric binding site. Overall, our data point to a role of PBP4 in PG cleavage during the longitudinal cell division and to a PG that might have been adapted to the symbiotic lifestyle.

Pharmacological and non-pharmacological therapies are simultaneously prescribed when treating hypertensive individuals with elevated cardiovascular risk (ie, metabolic syndrome individuals). However, it is unknown if the interactions between antihypertensive medication (AHM) and lifestyle interventions (ie, exercise training) may result in a better ambulatory blood pressure (ABP) control. To test this hypothesis, 36 hypertensive individuals with metabolic syndrome (MetS) under long-term prescription with AHM targeting the renin-angiotensin-aldosterone system (RAAS) were recruited. Before and after 4 months of high-intensity interval training (HIIT), participants completed two trials in a double-blind, randomized order: (a) placebo trial consisting of AHM withdrawal for 3 days and (b) AHM trial where individuals held their habitual dose of AHM. In each trial, 24-h mean arterial pressure (MAP) was monitored and considered the primary study outcome. Secondary outcomes included plasma renin activity (PRA) and aldosterone concentration to confirm withdrawal effects on RAAS, along with the analysis of urine albumin-to-creatinine ratio (UACR) to assess kidney function. The results showed main effects from AHM and HIIT reducing 24-h MAP (-5.7 mmHg, p < 0.001 and -2.3 mmHg, p = 0.007, respectively). However, there was not interaction between AHM and HIIT on 24-h MAP (p = 0.240). There was a main effect of AHM increasing PRA (p < 0.001) but no effect on plasma aldosterone concentration (p = 0.368). HIIT did not significantly improve RAAS hormones or the UACR. In conclusion, AHM and HIIT have independent and additive effects in lowering ABP. These findings support the combination of habitual AHM with exercise training with the goal to reduce ABP in hypertensive MetS individuals.

Adaptation to shifting temperatures is crucial for the survival of the bacterial pathogen Vibrio cholerae. Here, we show that colony rugosity, a biofilm-associated phenotype, is regulated by temperature in V. cholerae strains that naturally lack the master biofilm transcriptional regulator HapR. Using transposon-insertion mutagenesis, we found the V. cholerae ortholog of BipA, a conserved ribosome-associated GTPase, is critical for this temperature-dependent phenomenon. Proteomic analyses revealed that loss of BipA alters the synthesis of >300 proteins in V. cholerae at 22°C, increasing the production of biofilm-related proteins including the key transcriptional activators VpsR and VpsT, as well as proteins important for diverse cellular processes. At low temperatures, BipA protein levels increase and are required for optimal ribosome assembly in V. cholerae, suggesting that control of BipA abundance is a mechanism by which bacteria can remodel their proteomes. Our study reveals a remarkable new facet of V. cholerae’s complex biofilm regulatory network.

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor &beta, (TGF&beta, ). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strategy.

Ageratina pichinchensis (Kunth) is a plant used in traditional Mexican medicine to treat multiple ailments. However, there have not been biotechnological studies on producing compounds in in vitro cultures. The aim of this study was to establish a cell suspension culture of A. pichinchensis, quantify the anti-inflammatory constituents 2,3-dihydrobenzofuran (2) and 3-epilupeol (3), evaluate the anti-inflammatory potential of its extracts, and perform a phytochemical analysis. Cell suspension cultures were established in a MS culture medium of 30-g L&minus, 1 sucrose, 1.0-mg L&minus, 1 &alpha, naphthaleneacetic acid, and 0.1-mg L&minus, 1 6-furfurylaminopurine. The ethyl acetate extract of the cell culture analyzed by gas chromatography (GC) revealed that the maximum production of anti-inflammatory compounds 2 and 3 occurs on days eight and 16, respectively, improving the time and previously reported yields in callus cultures. The anti-inflammatory activity of these extracts exhibited a significant inhibition of nitric oxide (NO) production. Furthermore, a phytochemical study of the ethyl acetate (EtOAc) and methanol (MeOH) extracts from day 20 led to the identification of 17 known compounds. The structures of the compounds were assigned by an analysis of 1D and 2D NMR data and the remainder by GC&ndash, MS. This is the first report of the production of (-)-Artemesinol, (-)-Artemesinol glucoside, encecalin, and 3,5-diprenyl-acetophenone by a cell suspension culture of A. pichinchensis.

Many species of the Asteraceae family are used in traditional Mexican medicine for possessing healing properties. Ageratina pichinchensis (Asteraceae) is a plant used for the treatment of gastric ulcers, deep wounds and for its antifungal effects. The aim of this study was to establish a cell suspension culture of A. pichinchensis, quantify the anti-inflammatory constituents 2,3-dihydrobenzofuran and 3-epilupeol, to evaluate the anti-inflammatory potential of its extracts and perform a phytochemical analysis. Cell suspension cultures were established in MS culture medium supplemented with 30 g L-1 sucrose and 1.0 g L-1 α-naphthaleneacetic acid (NAA) plus 0.1 mg L-1 6-furfurylaminopurine (KIN). The ethyl acetate extracts of cell suspension cultures analyzed by GC revealed that the maximum production of compounds The anti-inflammatory activity of these extracts exhibited significant inhibition of NO production. Furthermore, the phytochemical study of EtOAc and MeOH extracts of the biomass on day 20 led to the identification of 17 known compounds. The structures of compounds were assigned by analysis of 1D and 2D NMR data and the remainder by GC-MS. This is the first report of the production of the (-)-Artemesinol, (-)-Artemesinol glucoside, Encecalin and 3,5-diprenyl-acetophenone compounds by a cell suspension cultures of A. pichinchensis.

The bacterial cell wall is composed primarily of peptidoglycan (PG), a poly-aminosugar that is essential to sustain cell shape, growth and structural integrity. PG is synthesized by two different types of synthase complexes (class A Penicillin-binding Proteins [PBP]s/Lpos and Shape, Elongation, Division, Sporulation [SEDS]/class B PBP pairs) and degraded by ‘autolytic’ enzymes to accommodate growth processes. It is thought that autolsyin activity (and particulary the activity of endopeptidases, EPs) is required for PG synthesis and incorporation by creating gaps that are patched and paved by PG synthases, but the exact relationship between autolysins and the separate synthesis machineries remains incompletely understood. Here, we have probed the consequences of EP depletion for PG synthesis in the diarrheal pathogen Vibrio cholerae. We found that EP depletion resulted in severe morphological defects, increased cell mass, a decline in viability, and continuing (yet aberrant) incorporation of cell wall material. Mass increase and cell wall incorporation proceeded in the presence of Rod system inhibitors, but was abolished upon inhibition of aPBPs. However, the Rod system remained functional (i.e., exhibited sustained directed motion) even after prolonged EP depletion, without effectively promoting cell elongation. Lastly, heterologous expression of an EP from Neisseria gonorrhoeae could fully complement growth and morphology of an EP-insufficient V. cholerae. Overall, our findings suggest that in V. cholerae, the Rod system requires endopeptidase activity (but not necessarily direct interaction with EPs) to promote cell expansion and substantial PG incorporation, whereas aPBPs are able to engage in sacculus construction even during severe EP insufficiency.ImportanceSynthesis and turnover of the bacterial cell wall must be tightly co-ordinated to avoid structural integrity failure and cell death. Details of this coordination are poorly understood, particularly if and how cell wall turnover enzymes are required for the activity of the different cell wall synthesis machines. Our results suggest that in Vibrio cholerae, one class of turnover enzymes, the endopeptidases, are required only for substantial PG incorporation mediated by the Rod system, while the aPBPs maintain structural integrity during endopeptidase insufficiency. Our results suggest that aPBPs are more versatile than the Rod system in their ability to recognize cell wall gaps formed by autolysins other than the major endopeptidases, adding to our understanding of the co-ordination between autolysins and cell wall synthases. A detailed understanding of autolysin biology may promote the development of antibiotics that target these essential turnover processes.

33 p. ATP-binding cassette (ABCG2) is an efflux transporter that extrudes xenotoxins from cells in liver, intestine, mammary gland, brain and other organs, affecting the pharmacokinetics, brain accumulation and secretion into milk of several compounds, including antitumoral, antimicrobial and anti-inflammatory drugs. The aim of this study was to investigate whether the widely used anti-inflammatory drug meloxicam is an Abcg2 sustrate, and how this transporter affects its systemic distribution. Using polarized ABCG2-transduced cell lines, we found that meloxicam is efficiently transported by murine Abcg2 and human ABCG2. After oral administration of meloxicam, the area under the plasma concentration-time curve in Abcg2-/- mice was 2-fold higher than in wild type mice (146.06 ± 10.57 μg·h/ml versus 73.80 ± 10.00 μg·h/ml). Differences in meloxicam distribution were reported for several tissues, with a 20-fold higher concentration in the brain of Abcg2-/- compared to wild-type mice. Meloxicam secretion into milk was also affected by the transporter, with a 2.5-fold higher milk-to-plasma ratio in wild-type compared with Abcg2-/- lactating female mice (0.58 ± 0.08 versus 0.23 ± 0.06). We conclude that Abcg2 is an important determinant of the plasma and brain distribution of meloxicam and is clearly involved in its secretion into milk. This study was supported by the research projects AGL2015-65626-R (MINECO/FEDER, UE) and RTI2018-100903-B-I00 (AEI/FEDER, UE); and by the predoctoral grants from the Ministry of Economy, Industry and Competitiveness (BES-2016-077235 grant to AMGL), and from Spanish Ministry of Education, Culture and Sport (FPU14/05131 grant to DGM AND FPU18/01559 grant to EBP); and the Junta de Castilla y Leon and European Regional Development Fund (Post-Doctoral Fellowship LE011P17 grant to IAF).

Peptidoglycan (PG) is an essential cell wall component that maintains the morphology and viability of nearly all bacteria. Its biosynthesis requires periplasmic transpeptidation reactions which construct peptide cross-linkages between polysaccharide chains to endow mechanical strength. However, tracking transpeptidation reaction in vivo and in vitro is challenging, mainly due to the lack of efficient, biocompatible probes. Here, we report the design, synthesis, and application of rotor-fluorogenic D-amino acids (RfDAAs) enabling real-time, continuous tracking of transpeptidation reactions. These probes enable monitoring PG biosynthesis in real time through visualizing transpeptidase reactions in live cells, as well as real-time activity assays of D,D-, L,D-transpeptidases, and sortases in vitro. The unique ability of RfDAAs to become fluorescent when incorporated into PG provides a powerful new tool to study PG biosynthesis with high temporal resolution and prospectively enable high-throughput screening for inhibitors of PG biosynthesis., Graphical Abstract

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over; the latter process is mediated by PG cleavage enzymes, for example, the endopeptidases (EPs). EPs themselves are essential for growth but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulated in vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogen Vibrio cholerae . Our data suggest that ShyA assumes two drastically different conformations: a more open form that allows for substrate binding and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activity in vitro and in vivo, and these results were recapitulated in EPs from the divergent pathogens Neisseria gonorrheae and Escherichia coli . Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain 1 from the M23 active site, likely through conformational rearrangement in vivo.

Cell movements are essential for morphogenesis during animal development. Epiboly is the first morphogenetic process in zebrafish in which cells move en masse to thin and spread the deep and enveloping cell layers of the blastoderm over the yolk cell. While epiboly has been shown to be controlled by complex molecular networks, the contribution of reactive oxygen species (ROS) to this process has not previously been studied. Here, we show that ROS are required for epiboly in zebrafish. Visualization of ROS in whole embryos revealed dynamic patterns during epiboly progression. Significantly, inhibition of NADPH oxidase activity leads to a decrease in ROS formation, delays epiboly, alters E-cadherin and cytoskeleton patterns and, by 24 h post-fertilization, decreases embryo survival, effects that are rescued by hydrogen peroxide treatment. Our findings suggest that a delicate ROS balance is required during early development and that disruption of that balance interferes with cell adhesion, leading to defective cell motility and epiboly progression.

Eleven 4′-alkoxy chalcones were synthesized and biologically evaluated for their antiproliferative activity against four human tumor cell lines (PC-3, MCF-7, HF-6, and CaSki). Compounds 3a-3d and 3f were selective against PC-3, with IC50 values ranging from 8.08 to 13.75 μM. In addition, chalcones 3a-3c did not affect the normal fibroblasts BJ cells. The most active and selective compounds were further evaluated for their effect on the progression of cell cycle in PC-3 cells, and chalcones 3a and 3c induced a G2/M phase arrest. Furthermore, it was found that these three chalcones induced the mitochondrial apoptotic pathway by regulating Bax and Bcl-2 transcripts and by increasing caspase 3/7 activation. Otherwise, the QSAR model indicates that the double bond of the α,β-unsaturated carbonyl, as well as the planar structure geometry, are important to the biological activity of the synthetized chalcones. Based on these studies, it was concluded that withdrawing substituents in ring A, decrease the antiproliferative activity. This is related to the possible mechanism of action of these compounds, where a Michael addition needs to take place in order to be a potent anticancer agent.

In many Thermus thermophilus strains, nitrate respiration is encoded in mobile genetic regions, along with regulatory circuits that modulate its expression based on anoxia and nitrate presence. The ...

Denitrifying bacteria carry out nitrate and nitrite respiration inside and outside the cell, respectively. In Thermus thermophilus, nitrate and nitrite transport processes are carried out by major facilitator superfamily (MFS) transporters. The sequence of the nar operon of nitrate-only respiring strains of T. thermophilus includes two tandemly organized MFS transporter genes (narK and narT) of the NarK1 and NarK2 families. Both can function as nitrate/nitrite antiporters, but NarK has been proposed as more specific for nitrate whereas NarT more specific for nitrite. In some nitrate- and nitrite-respiring strains of the same species, a single MFS transporter (NarO) belonging to a different MFS subfamily appears. To analyze the role of this single MFS in the same genetic context, we transferred the two types of nar operon to the aerobic strain HB27, and further included in both of them the ability to respire nitrite. The new denitrifying strains HB27dn, with two MFS, and HB27dp, with a single one, were used to isolate mutants devoid of transporters. Through in trans complementation experiments, we demonstrate that the NarO single MFS works efficiently in the transport of both nitrate and nitrite., This work was supported by grant BIO2016-77031-R from the Spanish Ministry of Economy and Competitiveness. An institutional grant from Fundación Ramón Areces to the CBMSO is also acknowledged

In this study the biosurfactant (Bs) production of two Serratia marcescens strains (SM3 and its isogenic SMRG-5 strain) was improved and the tenso-active agents were purified and characterized. A 23 factorial design was used to evaluate the effect of nitrogen and carbon sources on the surface tension (ST) reduction and emulsion index (EI24) of the produced Bs. Optimum Bs production by SM3 was achieved at high concentrations of carbon and nitrogen, reducing ST to 26.5 ± 0.28 dynes/cm, with an EI24 of 79.9 ± 0.2 %. Meanwhile, the best results for SMRG-5 were obtained at low concentrations, reducing the ST to 25.2 ± 0.2 dynes/cm, with an EI24 of 89.7 ± 0.28 %. The optimal conditions for Bs production were scaled up in a 2-L reactor, yielding 4.8 and 5.2 g/L for SM3 and SMRG-5, respectively. GC-MS analysis revealed the presence of two different lipopeptides (octadecanoic and hexadecanoic acid for SM3 and SMRG5, respectively). Both strains were capable of benzo [a] pyrene removal (59 % after 72 h of culture). This article is protected by copyright. All rights reserved

Background A key process in cell regulation is protein phosphorylation, which is catalyzed by protein kinases and phosphatases. However, phosphoproteomics studies are difficult because of the complexity of protein phosphorylation and the number of phosphorylation sites. Methods We describe an efficient approach analyzing phosphopeptides in single, separated protein by two-dimensional gel electrophoresis. In this method, a titanium oxide (TiO2)-packed NuTip is used as a phosphopeptide trap, together with displacers as lactic acid in the loading buffer to increase the efficiency of the interaction between TiO2 and phosphorylated peptides. Results The results were obtained from the comparison of mass spectra of proteolytic peptides of proteins with a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) instrument. Conclusions This method has been applied to identifying phosphoproteins involved in the symbiosis Rhizobium etli-Phaseolus vulgaris.

Glucose 6 phosphate dehydrogenase (G6PD) is called the house keeping enzyme essential for all cells survival. G6PD deficiency is best studied for its role in hemolysis as this enzyme is the only source of nicotinamide adenine dinucleotide phosphate (NADPH) in red blood cells (RBCs) and is the cells defense against reactive oxygen species. We present one of the rare complications of G6PD deficiency in this case where all other causes of priapism were ruled out. J Med Cases. 2019;10(9):271-273 doi: https://doi.org/10.14740/jmc3355

Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, ΔcdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, α-ketoglutarate, ATP, NAD+, UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that ΔcdnL cells are glutamate auxotrophs, and ΔcdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites., Author summary To grow and divide, bacteria must accumulate precursor molecules to support duplication and expansion of cellular materials. One mechanism by which bacteria do this is by regulating the expression of genes whose products are important for production of these molecules. How gene expression is maintained or altered to support synthesis of appropriate molecules to balance growth with nutrient availability is not fully understood. In this paper, we describe the role of a regulator of gene expression called CdnL in maintaining levels of molecules required for bacterial growth and reproduction. CdnL broadly impacts the levels of genes required for most biosynthetic processes. CdnL’s broad impact on transcription has downstream consequences on growth rate, cell shape, and nutrient requirements for growth. We report that CdnL is particularly important for maintaining levels of the amino acid glutamate and the cell wall precursor lipid II, each of which is critical for supporting proper growth and cell morphology. Our results implicate CdnL as a broadly conserved regulator of metabolic homeostasis, growth, and cell shape in bacteria.

International audience; Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.

Bacteria are surrounded by a protective peptidoglycan cell wall. Provided that this structure and the enzymes involved are the preferred target for our most successful antibiotics, determining its structural and chemical complexity is of the highest interest. Traditionally, high-performance liquid chromatography (HPLC) analyses have been performed, but these methods are very time consuming in terms of sample preparation and chromatographic separation. Here we describe an optimized method for preparation of Gram-negative bacteria peptidoglycan and its subsequent analysis by ultra-performance liquid chromatography (UPLC). The use of UPLC in peptidoglycan analyses provides a dramatic reduction of the sample volume and hands-on time required and, furthermore, permits in-line mass spectrometry (MS) of the UPLC resolved muropeptides, thus facilitating their identification. This method improves our capability to perform high throughput analysis to better understand the cell-wall biology.

Traumatic spinal cord injury (TSCI) can cause paralysis and permanent disability. Rehabilitation (RB) is currently the only accepted treatment, although its beneficial effect is limited. The development of biomaterials has provided therapeutic possibilities for TSCI, where our research group previously showed that the plasma-synthesized polypyrrole/iodine (PPy/I), a biopolymer with different physicochemical characteristics than those of the PPy synthesized by conventional methods, promotes recovery of motor function after TSCI. The present study evaluated if the plasma-synthesized PPy/I applied in combination with RB could increase its beneficial effects and the mechanisms involved. Adult rats with TSCI were divided into no treatment (control); biopolymer (PPy/I); mixed RB by swimming and enriched environment (SW/EE); and combined treatment (PPy/I + SW/EE) groups. Eight weeks after TSCI, the general health of the animals that received any of the treatments was better than the control animals. Functional recovery evaluated by two scales was better and was achieved in less time with the PPy/I + SW/EE combination. All treatments significantly increased βIII-tubulin (nerve plasticity) expression, but only PPy/I increased GAP-43 (nerve regeneration) and MBP (myelination) expression when were analyzed by immunohistochemistry. The expression of GFAP (glial scar) decreased in treated groups when determined by histochemistry, while morphometric analysis showed that tissue was better preserved when PPy/I and PPy/I + SW/EE were administered. The application of PPy/I + SW/EE, promotes the preservation of nervous tissue, and the expression of molecules related to plasticity as βIII-tubulin, reduces the glial scar, improves general health and allows the recovery of motor function after TSCI. The implant of the biomaterial polypyrrole/iodine (PPy/I) synthesized by plasma (an unconventional synthesis method), in combination with a mixed rehabilitation scheme with swimming and enriched environment applied after a traumatic spinal cord injury, promotes expression of GAP-43 and βIII-tubulin (molecules related to plasticity and nerve regeneration) and reduces the expression of GFAP (molecule related to the formation of the glial scar). Both effects together allow the formation of nerve fibers, the reconnection of the spinal cord in the area of injury and the recovery of lost motor function. The figure shows the colocalization (yellow) of βIII-tubilin (red) and GAP-43 (green) in fibers crossing the epicenter of the injury (arrowheads) that reconnect the rostral and caudal ends of the injured spinal cord and allowed recovery of motor function.

Most bacteria surround themselves with a cell wall, a strong meshwork consisting primarily of the polymerized aminosugar peptidoglycan (PG). PG is essential for structural maintenance of bacterial cells, and thus for viability. PG is also constantly synthesized and turned over, the latter process is mediated by PG cleavage enzymes, for example the endopeptidases (EPs). EPs themselves are essential for growth, but also promote lethal cell wall degradation after exposure to antibiotics that inhibit PG synthases (e.g., β-lactams). Thus, EPs are attractive targets for novel antibiotics and their adjuvants. However, we have a poor understanding of how these enzymes are regulatedin vivo, depriving us of novel pathways for the development of such antibiotics. Here, we have solved crystal structures of the LysM/M23 family peptidase ShyA, the primary EP of the cholera pathogenVibrio cholerae.Our data suggest that ShyA assumes two drastically different conformations; a more open form that allows for substrate binding, and a closed form, which we predicted to be catalytically inactive. Mutations expected to promote the open conformation caused enhanced activityin vitroandin vivo, and these results were recapitulated in EPs from the divergent pathogensNeisseria gonorrheaeandEscherichia coli. Our results suggest that LysM/M23 EPs are regulated via release of the inhibitory Domain1 from the M23 active site, likely through conformational re-arrangementin vivo.SignificanceBacteria digest their cell wall following exposure to antibiotics like penicillin. The endopeptidases (EPs) are among the proteins that catalyze cell wall digestion processes after antibiotic exposure, but we do not understand how these enzymes are regulated during normal growth. Herein, we present the structure of the major EP from the diarrheal pathogenVibrio cholerae.Surprisingly, we find that EPs from this and other pathogens appear to be produced as a largely inactive precursor that undergoes a conformational shift exposing the active site to engage in cell wall digestion. These results enhance our understanding of how EPs are regulated and could open the door for the development of novel antibiotics that overactivate cell wall digestion processes.

Breast cancer is currently the leading cause of cancer death among women worldwide. AP-1 (c-Fos/c-Jun) isassociated with proliferation and survival, while cytoplasmic c-Fos activates phospholipid synthesis in cellsinduced to differentiate or grow. Estrogen receptor α 46 (ERα46) is a splice variant of full-length ERα66 and it isknown that it has an inhibitory role in cancer cell growth. We investigated c-Fos localization, its relationship toAP-1, the non genomic pathway of phospho-Tyr537-ERα66, as well as ERα46 and ERα66 isoforms in ratmammary gland development and carcinogenic transformation, and in mammary tumors. Female rats wereinjected: a) saline solution (Control mammary gland, CMG) or b) N-Nitroso-N-methyl urea (NMU), and sampleswere taken at 60, 90, 120 and 150 days of life. In addition, we analyzed hormone-dependent (HD) and in-dependent (HI) tumors in ovariectomized rats, and intact tumors (IT) in non-ovariectomized ones. Our resultsshow that, in CMG, nuclear c-Fos and proliferation decreased with age, AP-1 content was low, and nuclearERα46/ERα66 ratio was higher than 1. In NMU, nuclear c-Fos and proliferation increased with carcinogenictransformation, AP-1 content was high, and nuclear ERα46/ERα66 was below 1. As tumor grade increased,proliferation, nuclear c-Fos and AP-1 expression were negatively associated to nuclear ERα46/ERα66 in IT. InHD, nuclear ERα46/ERα66, nuclear c-Fos expression, AP-1 levels and proliferation were lower than in HI, whosegrowth is estrogen-independent. Phospho-Tyr537-ERα66 content and ERK1/2 activation were associated withAP-1 levels and cell proliferation. Collectively, our findings support the notion that variant detection andERα46/ERα66 ratio could shed light on the role of ERα isoforms in mammary gland transformation and thebehavior of ERα positive mammary tumors. Fil: Gutiérrez, Alicia Beatriz. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Sambuco, Lorena Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina Fil: Alvarez, Laura. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina Fil: Núñez, Mariel Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina Fil: Bergoc, Rosa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina Fil: Zotta, Elsa. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Ciencias Fisiológicas. Laboratorio de Fisiopatogenia; Argentina Fil: Martín, Gabriela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Físico Matemática; Argentina Fil: Randi, Andrea Silvana. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana. Cátedra de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Multidrug resistance (MDR) has become a major obstacle in the treatment of cancer, and is associated with mechanisms such as increased drug outflow, reduction of apoptosis, and/or altered drug metabolism. These problems can be mitigated by the coadministration of agents known as chemosensitizers, as they can reverse resistance to anticancer drugs and eventually resensitize cancer cells. We explore the chemosensitizing effect of Achillin, a guaianolide-type sesquiterpene lactone isolated from the Mexican medicinal plant Artemisia ludovisiana, to reverse MDR in Hep3B/PTX cells of hepatocellular carcinoma, which present resistance to paclitaxel (PTX). Achillin showed an important effect as chemosensitizer, indeed, the cytotoxic effect of PTX (25 nM) was enhanced, and the induction of G2/M phase cell cycle arrest and apoptosis were potentiated when combining with Achillin (100 &mu, M). In addition, we observed that Achillin decreases P-gp levels and increases the intracellular retention of doxorubicin in Hep3B/PTX cells, in addition, homology structural modeling and molecular docking calculations predicted that Achillin interacts in two regions (M-site and R-site) of transporter drug efflux P-glycoprotein (P-gp). Our results suggest that the chemosensitizer effect demonstrated for Achillin could be associated with P-gp modulation. This work also provides useful information for the development of new therapeutic agents from guaianolide-type sesquiterpene lactones like Achillin.

BolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster-containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur-containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins., BolA family proteins are conserved in Gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well described. Here, we investigated the roles of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogen’s morphology. Notably, the bizarre-shaped, multipolar, elongated, and wide cells that predominated in exponential-phase ΔibaG V. cholerae cultures were not observed in stationary-phase cultures. The V. cholerae ΔibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall-acting antibiotics and bile, and was defective in intestinal colonization. ΔibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutant’s morphological defects and sensitivity to envelope stressors. Transposon insertion sequencing analysis of ibaG’s genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, copurification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.

Bursera copallifera(DC) Bullock (Burseraceae) is a plant species used in folk medicine for the treatment of inflammation (bronchitis, cough). The dichloromethane-methanol extract (DML) of B. copallifera leaves was found to possess important antiproliferative and anti-inflammatory activities. In order to further correlate these activities with the phytochemical components, the bioactivity-guided fractionation of the DML extract allowed the isolation of an active fraction which was constituted of five compounds characterized as as luteolin-3'-O-(3''-O-E-p-coumaroyl)-α-L-rhamnopyranoside (1), luteolin-3'-O-(2''-O-E-p-coumaroyl)-α-L-rhamnopyranoside (2), a-amyrin (3), 3-epilupeol (4) and stigmasterol (5). Among the five isolated constituents, the triterpenes 3 and 4, and the sterol 5 have been already well known by their anti-inflammatory properties. Flavone 2 (Luteolin-3'-O-(2''-O-E-p-coumaroyl)-α-L-rhamnopyranoside)was responsible for the antiproliferative activity, showing in vitro cytotoxic activity against MCF-7 human cancer cell line with IC50 value of 13.9 µM. By the other hand flavone (1) exhibited COX-1 inhibition (IC50 = 93 mM). Flavone 1, together with the triterpenes 3 and 4 were the responsible for inhibiting TPA-induced inflammation by inhibit COX-1 enzyme. Therefore, these results support the traditional use of the infusion made with the leaves of B. copallifera as an anti-inflammatory agent.

Prolonging the clinical effectiveness of {beta}-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted D-cycloserine (DCS) transporter gene cycA gene re-sensitizes MRSA to {beta}-lactam antibiotics. The cycA mutation also resulted in hyper-susceptibility to DCS, an alanine analogue antibiotic that inhibits the alanine racemase and D-Alanine ligase enzymes required for D-Alanine incorporation into peptidoglycan. Amino acid transport studies showed that CycA functions as an alanine permease, and the cycA mutation was accompanied by increased accumulation of muropeptides with tripeptide stems lacking the terminal D-Ala-D-Ala, and reduced peptidoglycan cross linking. Exposure of MRSA to DCS was also associated with a dose-dependent accumulation of muropeptides with tripeptide stems and reduced PG crosslinking. Because impaired alanine transport or DCS-treatment have similar effects on PG structure, synergism between {beta}-lactams and DCS was investigated. Therapeutically achievable concentrations of DCS re-sensitised MRSA to {beta}-lactam antibiotics in vitro and significantly enhanced MRSA eradication in a mouse bacteraemia model. The susceptibility of several other ESKAPE group pathogens to {beta}-lactams was also increased by DCS. These data reveal the potential of DCS, or new drugs targeting CycA, to facilitate the reintroduction of {beta}-lactam antibiotics for the treatment of infections caused by MRSA.nnAuthor SummaryTreatment options for infections caused by ESKAPE group pathogens continue to dwindle with increasing antimicrobial resistance. Particularly important are the {beta}-lactams, which remain first line antibiotics for many infections, and finding new ways to maintain their clinical effectiveness is a priority. Here we report that mutation of cycA in MRSA, which was found to encode an alanine permease, was associated with hyper-susceptibility to {beta}-lactam antibiotics and D-cycloserine (DCS), used in the treatment of multi-drug resistant tuberculosis infections. DCS re-sensitised MRSA to oxacillin and other {beta}-lactams in vitro and significantly enhanced oxacillin activity in a mouse model of bacteraemia. DCS also sensitised other ESKAPE pathogens to {beta}-lactams revealing the therapeutic potential of DCS as an adjunct drug for these important antibiotics.

Breast cancer incidence is increasing globally and pesticides exposure may impact risk of developing this disease. Hexachlorobenzene (HCB) and chlorpyrifos (CPF) act as endocrine disruptors, inducing proliferation in breast cancer cells. Vascular endothelial growth factor-A (VEGF-A), cyclooxygenase-2 (COX-2) and nitric oxide (NO) are associated with angiogenesis. Our aim was to evaluate HCB and CPF action, both weak aryl hydrocarbon receptor (AhR) ligands, on angiogenesis in breast cancer models. We used: (1) in vivo xenograft model with MCF-7 cells, (2) in vitro breast cancer model with MCF-7, and (3) in vitro neovasculogenesis model with endothelial cells exposed to conditioned medium from MCF-7. Results show that HCB (3 mg/kg) and CPF (0.1 mg/kg) stimulated vascular density in the in vivo model. HCB and CPF low doses enhanced VEGF-A and COX-2 expression, accompanied by increased levels of nitric oxide synthases (NOS), and NO release in MCF-7. HCB and CPF high doses intensified VEGF-A and COX-2 levels but rendered different effects on NOS, however, both pesticides reduced NO production. Moreover, our data indicate that HCB and CPF-induced VEGF-A expression is mediated by estrogen receptor and NO, while the increase in COX-2 is through AhR and NO pathways in MCF-7. In conclusion, we demonstrate that HCB and CPF environmental concentrations stimulate angiogenic switch in vivo. Besides, pesticides induce VEGF-A and COX-2 expression, as well as NO production in MCF-7, promoting tubulogenesis in endothelial cells. These findings show that pesticide exposure could stimulate angiogenesis, a process that has been demonstrated to contribute to breast cancer progression.

Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor σ70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where σ70localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show thatCaulobacter crescentuscells lacking CdnL have severe morphological and growth defects. Specifically,ΔcdnLcells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes. Notably, we find thatΔcdnLcells are severely limited in glutamate synthesis, rendering them auxotrophic for that amino acid. Moreover,ΔcdnLcells produce low amounts of the cell wall precursor lipid II, andΔcdnLis synthetic lethal with other genetic perturbations that limit lipid II production.ΔcdnLcells also have aberrant localization of MreB and CtpS, cytoskeletal proteins required for maintaining proper cell width and curvature. Interestingly, the localization of CtpS is dependent on availability of CTP, which is predicted to be low inΔcdnLcells. Our findings implicate CdnL as a global regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and through metabolite-mediated changes in localization of cytoskeletal regulators of cell shape.

Bacteria encode a variety of adaptations that enable them to survive during zinc starvation, a condition which is encountered both in natural environments and inside the human host. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, we have identified a novel member of this zinc starvation response, a cell wall hydrolase that retains function and is conditionally essential for cell growth in low-zinc environments. Other Gram-negative bacteria contain homologs that appear to be under similar regulatory control. These findings are significant because they represent, to our knowledge, the first evidence that zinc homeostasis influences cell wall turnover. Anti-infective therapies commonly target the bacterial cell wall; therefore, an improved understanding of how the cell wall adapts to host-induced zinc starvation could lead to new antibiotic development. Such therapeutic interventions are required to combat the rising threat of drug-resistant infections., The cell wall is a strong, yet flexible, meshwork of peptidoglycan (PG) that gives a bacterium structural integrity. To accommodate a growing cell, the wall is remodeled by both PG synthesis and degradation. Vibrio cholerae encodes a group of three nearly identical zinc-dependent endopeptidases (EPs) that are predicted to hydrolyze PG to facilitate cell growth. Two of these (ShyA and ShyC) are conditionally essential housekeeping EPs, while the third (ShyB) is not expressed under standard laboratory conditions. To investigate the role of ShyB, we conducted a transposon screen to identify mutations that activate shyB transcription. We found that shyB is induced as part of the Zur-mediated zinc starvation response, a mode of regulation not previously reported for cell wall lytic enzymes. In vivo, ShyB alone was sufficient to sustain cell growth in low-zinc environments. In vitro, ShyB retained its d,d-endopeptidase activity against purified sacculi in the presence of the metal chelator EDTA at concentrations that inhibit ShyA and ShyC. This insensitivity to metal chelation is likely what enables ShyB to substitute for other EPs during zinc starvation. Our survey of transcriptomic data from diverse bacteria identified other candidate Zur-regulated EPs, suggesting that this adaptation to zinc starvation is employed by other Gram-negative bacteria.

By using a zebrafish embryo model to guide the chromatographic fractionation of antimitotic secondary metabolites, seven podophyllotoxin-type lignans were isolated from a hydroalcoholic extract obtained from the steam bark of Bursera fagaroides. The compounds were identified as podophyllotoxin (1), &beta, peltatin-A-methylether (2), 5&prime, desmethoxy-&beta, peltatin-A-methylether (3), desmethoxy-yatein (4), desoxypodophyllotoxin (5), burseranin (6), and acetyl podophyllotoxin (7). The biological effects on mitosis, cell migration, and microtubule cytoskeleton remodeling of lignans 1&ndash, 7 were further evaluated in zebrafish embryos by whole-mount immunolocalization of the mitotic marker phospho-histone H3 and by a tubulin antibody. We found that lignans 1, 2, 4, and 7 induced mitotic arrest, delayed cell migration, and disrupted the microtubule cytoskeleton in zebrafish embryos. Furthermore, microtubule cytoskeleton destabilization was observed also in PC3 cells, except for 7. Therefore, these results demonstrate that the cytotoxic activity of 1, 2, and 4 is mediated by their microtubule-destabilizing activity. In general, the in vivo and in vitro models here used displayed equivalent mitotic effects, which allows us to conclude that the zebrafish model can be a fast and cheap in vivo model that can be used to identify antimitotic natural products through bioassay-guided fractionation.

8 p.-6 fig., Paclitaxel (PTX) is currently used as a front-line chemotherapeutic agent for several types of cancer, including ovarian carcinoma; however, PTX-resistance frequently arises through multiple mechanisms. The development of new strategies using natural compounds and PTX in combination has been the aim of several prior studies, in order to enhance the efficacy of chemotherapy. In this study, we found the following: (i) gallic acid (GA), a phenolic compound, potentiated the capacity of PTX to decrease proliferation and to cause G2/M cycle arrest in the PTX-resistant A2780AD ovarian cancer cell line; (ii) GA exerted a pro-oxidant action by increasing the production of reactive oxygen species (ROS), and co-treatment with the antioxidant agent N‑acetyl-L‑cysteine (NAC) prevented GA+PTX-induced cell proliferation inhibition and G2/M phase arrest; (iii) PTX stimulated ERK phosphorylation/activation, and co-treatment with the MEK/ERK inhibitor PD98049 potentiated the proliferation inhibition and G2/M phase arrest; (iv) and finally, GA abrogated the PTX-induced stimulation of ERK phosphorylation, a response that was prevented by co-treatment with NAC. Taken together, these results indicate that GA sensitizes PTX-resistant ovarian carcinoma cells via the ROS‑mediated inactivation of ERK, and suggest that GA could represent a useful co-adjuvant to PTX in ovarian carcinoma treatment., Jessica Nallely Sánchez-Carranza acknowledges fellowship 351450 from CONACYT and the support from CONACYT (CB240801, LN 279905), CONACYT-FOMIX (224038). Partial support from UAEM (Grants SI-DGDI-UAEM/13/289) is acknowledged.

Three new aryldihydronaphthalene-type lignans, namely, 7′,8′-dehydropodophyllotoxin (1); 7′,8′-dehydro acetylpodophyllotoxin (2); and 7′,8′-dehydro trans-p-cumaroylpodophyllotoxin (3), were isolated from the stem bark of Bursera fagaroides var. fagaroides (Burseraceae), together with six known lignans, podophyllotoxin (4), acetylpodophyllotoxin (5), 5′-desmethoxy-β-peltatin A methylether (6), acetylpicropodophyllotoxin (7), burseranin (8), and hinokinin (9). The coumarin scopoletin (10) was also isolated from this bark. The chemical structure of all these compounds was determined by spectroscopic analyses including 2D NMR. We demonstrated that compounds 1–3 show different degrees of cytotoxic activity against human nasopharyngeal (KB), colon (HF-6), breast (MCF-7) and prostate (PC-3) cancer cell lines, with IC50 values ranging from 1.49 to 1.0 × 10−5 μM. In vivo studies of the effect of these natural lignans on the cell cycle, cell migration and microtubule cytoskeleton of developing zebrafish embryos, demonstrated their antimitotic molecular activity by disturbing tubulin. This is the first report on the occurrence of aryldihydronaphthalene lignans in the genus Bursera of the Burseraceae family, as well as on the determination of their cytotoxic activity and mechanism of action.

In Mexico, the oil seed from Calophyllum brasiliense is traditionally used for lighting, but the oil seeds and leaves have not yet been evaluated. In this work, two sites of C. brasiliense were studied: San Andres Tuxtla and Pajapan. The aim of this study was to characterize the fatty acid profile of the seed oil, leaves, and callus cultures from leaves or seed explants. Fatty acids of the leaves, seeds, and callus culture samples were extracted with hexane, esterified, and then analyzed in a Gas chromatography–mass spectrometry. The San Andres Tuxtla seeds had a significantly higher (P ≤ 0.05) oil content: 58.2% versus 47.6% in Pajapan seeds. The seed oil recovered from the San Andres Tuxtla samples showed higher amounts of linoleic acid (41.3%), while the oil recovered from the Pajapan seeds had remarkable oleic acid content (39.3%). The physicochemical parameters of San Andres Tuxtla and Pajapan oils such as the density, viscosity, iodine, acid, and saponification values were measured and met the industrial standards for oil feedstock for biodiesel. The biomass growth of callus cultures was enhanced, and callus could produce fatty acids. These were mainly total saturated acids such as palmitic acid (31.32–39.18%) followed by stearic acid (19.52–21.18%). The main unsaturated fatty acid was oleic acid with values ranging from 23.14 to 31.63%. This is the first report on the fatty acid composition of seeds, leaves, and callus cultures from C. brasiliense. Therefore, this study can help to promote the importance of this agricultural product and can lead to biotechnological tools for the sustainable production of fatty acids or secondary metabolites.

A comprehensive study into the potential of bioprocessing techniques (sourdough fermentation and technological aids) for improving the technological, sensory, and nutritional properties of breads made using brewer's spent grain (BSG) was undertaken. Xylanase and dough conditioner altered the mixing and pasting properties of the flours, improved the specific volume and texture of breads and delayed staling in BSG breads when added to both sourdough and non-sourdough BSG breads. The aromatic properties were determined by volatile analysis and were influenced by sourdough fermentation. Ferulic and 4-coumaric acids were the main phenolic acids found in insoluble bound form in BSG flour, while the phenolic profile was different for the free extracts. Sourdough fermentation and the use of enzymes increased the antioxidant capacity of breads.

In the environment bacteria share their habitat with a great diversity of organisms, from microbes to humans, animals and plants. In these complex communities, the production of extracellular effectors is a common strategy to control the biodiversity by interfering with the growth and/or viability of nearby microbes. One of such effectors relies on the production and release of extracellular D-amino acids which regulate diverse cellular processes such as cell wall biogenesis, biofilm integrity, and spore germination. Non-canonical D-amino acids are mainly produced by broad spectrum racemases (Bsr). Bsr's promiscuity allows it to generate high concentrations of D-amino acids in environments with variable compositions of L-amino acids. However, it was not clear until recent whether these molecules exhibit divergent functions. Here we review the distinctive biological roles of D-amino acids, their mechanisms of action and their modulatory properties of the biodiversity of complex eco-systems.

Bacteria live in polymicrobial communities under tough competition. To persist in a specific niche many species produce toxic extracellular effectors as a strategy to interfere with the growth of nearby microbes. One of such effectors are the non-canonical D-amino acids. Here we describe a method to test the effect of D-amino acid production in fitness/survival of bacterial subpopulations within a community. Co-cultivation methods usually involve the growth of the competing bacteria in the same container. Therefore, within such mixed cultures the effect on growth caused by extracellular metabolites cannot be distinguished from direct physical interactions between species (e.g., T6SS effectors). However, this problem can be easily solved by using a filtration unit that allows free diffusion of small metabolites, like L- and D-amino acids, while keeping the different subpopulations in independent compartments. With this method, we have demonstrated that D-arginine is a bactericide effector produced by Vibrio cholerae, which strongly influences survival of diverse microbial subpopulations. Moreover, D-arginine can be used as a cooperative instrument in mixed Vibrio communities to protect non-producing members from competing bacteria.