[Application and optimization of CRISPRi to the biology of Mycobacterium tuberculosis].

Tuberculosis, caused by infection with Mycobacterium tuberculosis (MTB), remains a global public health challenge. Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains make tuberculosis more difficult to control. New tools to study the biology of MTB can identify novel targets for drug discovery. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats interference (CRISPRi) combined with next-generation sequencing has provided many novel insights into the physiology and genetics of MTB. This review summarizes the application and optimization of CRISPRi in MTB biology.

Inhibition of Salmonalla enterica by a novel strain of Bacillus subtilis isolated from the faeces of healthy chickens.

1. The objective of the study was to demonstrate that Bacillus subtilis strain VL28 (BS-VL28), a novel strain isolated from faeces of healthy chicken, has potential as a probiotic.2. The study evaluated the probiotic properties of BS-VL28 and the effects of dietary supplementation of this strain on growth performance and mortality in chickens challenged with Salmonella enterica CT01.3. BS-VL28 exhibited a specific inhibitory activity against Escherichia coli CT11, Salmonella enterica CT01, Staphylococcus spp. CT21 and Streptococcus spp. CT31.4. BS-VL28 also showed an auto-aggregation percentage of 82%, co-aggregation activity greater than 60%, high tolerance to low pH (<2.0) under the presence of 0.05% bile salts. However, the results from the antibiotic susceptibility tests demonstrated that this strain was sensitive to erythromycin, gentamycin, doxycycline, norfloxacin, oxytetracycline, trimethoprim-sulfamethoxazole, enrofloxacin but was intermediate to neomycin.5. Inclusion of probiotic (5 g BS-VL28 (107 CFU/g) per kg of feed to diet of challenged chickens showed better performance and feed conversion rates (FCR). There was a significant decrease (p < 0.05) in mortality in the probiotic-treated group compared to the control and antibiotic-treated groups.6. From these results, BS-VL28 could potentially function as a probiotic for broilers.

Influences of copper-potassium ion exchange process on the optical bandgaps and spectroscopic properties of Cr3+/Yb3+ co-doped in lanthanum aluminosilicate glasses.

In this study, lanthanum aluminosilicate glasses with compositions of 45SiO2-20Al2O3-12.5LaF3-10BaF2-9K2O-1Cr2O3-2.5Yb2O3 (SALBK) were prepared using the conventional melting method and copper-potassium ion exchange process. Influences of the ion exchange process between copper and potassium on the visible, upconversion, and near-infrared luminescence spectra of Cr3+/Yb3+ co-doped under excitations of 343, 490, and 980 nm LD were investigated. The EDS analysis of SALBK glasses was measured to confirm the presence of atoms in the glasses. The values of direct and indirect bandgaps of Cr3+/Yb3+ co-doped SALBK glasses were calculated and analyzed. Besides, the energy exchange processes between Cu+, Cu2+ ions, and Cr3+, Yb3+ ions were also proposed and discussed.

Seizure threshold manipulation in electroconvulsive therapy via repetitive transcranial magnetic stimulation. A novel way of augmentation?

INTRODUCTION: A high seizure threshold (ST) is an impeding factor in certain patients, potentially preventing a successful electroconvulsive therapy (ECT) treatment. Several pharmacological and non-pharmacological methods have been put forward to augment ECT in such patients, however, to this date, only a handful of case reports existed about the potential role of repetitive transcranial magnetic stimulation (rTMS), as an augmentation method. OBJECTIVES: and Methods: In this randomized, double-blinded, sham controlled study, we set out to test the hypothesis of whether the application of high frequency transcranial magnetic stimulation (HF rTMS) lowers the seizure threshold for electroconvulsive therapy and whether it has an effect on other aspects of ECT treatment, such as seizure duration (SD), efficacy and safety. RESULTS: 46 patients treated for a major depressive episode, indicated for ECT, were recruited to this study. A significantly lower seizure threshold was observed in the experimental group during ECT titration, on average a decrease by 34.55%, from 34.23 mC to 22.4 mC, p < 0.001 (Wilcox test). We had not observed a significant effect of TMS stimulation before ECT on seizure duration or clinical outcome. Another potentially important observation of this study is that 4 patients in the experimental group developed transient symptoms of hypomania/mania, all of which were stabilized after the combined stimulation protocol was halted spontaneously within a week, without the need to administer mood stabilizers. CONCLUSION: It is likely that HF rTMS stimulation prior to ECT is a novel and simple way of reducing the ST, which is useful in certain groups of patients undergoing this important treatment modality.

[The State of The Jak/Stat Pathway Affects the Sensitivity of TumorCells to Oncolytic Enteroviruses].

A test of the sensitivity of seven colon cancer cell lines to a panel of 12 nonpathogenic human enteroviruses revealed significant differences in the ability of tumor cells to become infected and replicate different viral strains. Among the factors that can affect the sensitivity of cells to viruses are differences in the state of the mechanisms of antiviral protection, associated with a reaction to type I interferons. Using the two colon cancer cell lines CaCo2 and LIM1215 as a model, significant differences were revealed in the ability of cells to defend themselves against virus infection after 16 hours of treatment with 1000 units/mL of interferon-alpha. To study the effect of the state of the interferon response system, represented by the Jak/STAT signaling pathway, on the sensitivity of cells to different strains of enteroviruses, HEK293T cell lines were used. These are capable of supporting replication of each of the tested enteroviruses, as well as maintaining the ability to protect against viral infection after the treatment with interferon. Using the CRISPR/Cas9 system, HEK293T sublines with knockouts of the IFNAR1 and STAT2 genes were obtained. The sensitivity of control and knockout cells to infection with five strains of enteroviruses and the vesicular stomatitis virus was analyzed. It was noted that knockout of the IFNAR1 and STAT2 genes resulted in an increased sensitivity to all tested viruses. In knockout cells, the levels of reproduction of the vaccine derived of poliovirus type 1, Echoviruses 7 and 30, and Coxsackie viruses B5 and A7 were also significantly increased in comparison with the control HEK293T cells. Thus, deficiencies in the Jak/STAT signaling pathway in tumor cells lead to an overall increase in the sensitivity to oncolytic viruses.

Refractive index of nanoconfined water reveals its anomalous physical properties.

Despite extensive studies on the distinctive properties of water confined in a nanospace, the underlying mechanism and significance of the lengthscale involved in the confinement effects are still subjects of controversy. The dielectric constant and the refractive index in particular are key parameters in modeling and understanding nanoconfined water, yet experimental evidence is lacking. We report the measurement of the refractive indices of water in 10-100 nm spaces by exploiting the confinement of water and localized surface plasmons in a physicochemically well-defined nanocavity. The results revealed significantly low values and the scaling behavior of the out-of-plane refractive index n⊥ of confined water. They are attributed to the polarization suppression at the interfaces and the long-range correlation in electronic polarization facilitated by the strengthened H-bonding network. Using the refractive index as a sensing probe, we also observed anomalous stability of water structures over a wide range of temperature. Our measurement results provide essential feedback information for benchmarking water models and molecular interactions under nanoconfinement. This study also opens up a new methodology of using plasmon resonance in characterizing nanoconfined molecules and chemical reactions, and thus gives us fundamental insight into confinement effects.

Description and phylogenetic analyses of ribosomal transcription units from species of Fasciolidae (Platyhelminthes: Digenea).

Many members of Fasciolidae are common trematodes in cattle, buffaloes, sheep, elephants, pigs, with some capable of infecting humans also. In this study, the complete or near-complete sequences of ribosomal transcription unit (rTU or rDNA), each of Fasciola hepatica (Australia), Fascioloides jacksoni (Sri Lanka), Fasciolopsis buski (Vietnam) and three isolates of F. gigantica (Vietnam), were obtained and characterized. The full length of rDNA for each F. hepatica, 'hybrid' Fasciola sp., Fas. jacksoni and Fa. Buski, was 7657 bp, 7966 bp, 7781 bp and 8361 bp, with the complete intergenic spacer region (IGS) (862 bp, 1170 bp, 987 bp and 561 bp), respectively. The rDNA of two 'pure' F. gigantica isolates from Vietnam was 6794 bp with unsequenced IGS. For 28S rRNA genes the Fasciola spp. are equal, 1958 bp for 18S, 160 bp for 5.8S, 3863 bp and 454 bp for ITS1 but ITS2 differ by one nucleotide (Thymine) (359 or 360 bp). The ITS1 of the sensu lato Fa. buski has some distinguishable features, 286 bp for ITS2, 3862 bp for 28S and four repeat units of 356-361 bp each found in ITS1. The 28S rDNA analysis showed the lowest level of divergence (0-0.57%) between F. hepatica and F. gigantica and higher (2.23-2.62%) and highest (6-6.42%) for Fas. jacksoni and Fasciolopsis, respectively. The tree of 43 strains/species clearly produced a well-supported phylogeny, where 18 fasciolids consistently grouped, forming a discrete Fasciolidae clade, distinct from Philophthalmidae, Echinostomatidae and Echinochasmidae in Echinostomatoidea. Fascioloides jacksoni is outside Fasciola spp.: basal with Fas. magna, as previously demonstrated.

Functional outcome and muscle wasting in adults with tetanus.

BACKGROUND: In many countries, in-hospital survival from tetanus is increasing, but long-term outcome is unknown. In high-income settings, critical illness is associated with muscle wasting and poor functional outcome, but there are few data from resource-limited settings. In this study we aimed to assess muscle wasting and long-term functional outcome in adults with tetanus. METHODS: In a prospective observational study involving 80 adults with tetanus, sequential rectus femoris ultrasound measurements were made at admission, 7 days, 14 days and hospital discharge. Functional outcome was assessed at hospital discharge using the Timed Up and Go test, Clinical Frailty Score, Barthel Index and RAND 36-item Short Form Health Survey (SF-36) and 3 and 6 months after discharge using the SF-36 and Barthel Index. RESULTS: Significant muscle wasting occurred between hospital admission and discharge (p<0.01), particularly in severe disease, where a median 23.49% (interquartile range 10.01-26.07) reduction in rectus femoris cross-sectional area occurred in those with severe (Ablett grades 3 and 4) disease. Muscle mass at discharge was related to objective and subjective measures of physical and emotional function at discharge and 3 and 6 months after discharge. In patients >70 y of age, functional recovery at 6 months was reduced compared with younger patients. Hospital-acquired infection and age were risk factors for muscle wasting. CONCLUSIONS: Significant muscle wasting during hospitalization occurred in patients with tetanus, the extent of which correlates with functional outcome.

Molecular characterization of Fasciola jacksoni from wild elephants (Elephas maximus maximus) of Sri Lanka: a taxonomic evaluation.

Fasciola jacksoni is a significant contributor to the health and mortality of Asian elephants, particularly those in Sri Lanka. Despite the impact of fascioliasis on elephant populations, it is a neglected veterinary disease with limited taxonomic understanding. Molecular characterization and phylogenetic analysis of F. jacksoni were carried out to evaluate its suggested basal position in the Fasciolidae. Adult worms were collected during post-mortem of elephants, and eggs were collected from living elephants in National parks across Sri Lanka. Using the mitochondrial genes nicotinamide dehydrogenase subunit 1 (nad1) and cytochrome oxidase subunit 1 (cox1), and a partial 28S ribosomal DNA (28S rDNA), DNA sequences were generated from the F. jacksoni adult and egg material. Maximum likelihood (ML) phylogenetic analyses did not resolve F. jacksoni to be basal to the Fasciolidae. Furthermore, the ML analyses showed that the genus Fasciola was not monophyletic and that F. jacksoni was a sister species to the deer liver fluke Fascioloides magna. A clear framework is required to determine the taxonomic status of F. jacksoni and this current study provides the first detailed application of molecular techniques from multiple hosts across Sri Lanka with the production of reference DNA sequences for this important parasite.

Pretreatment Cerebrospinal Fluid Bacterial Load Correlates With Inflammatory Response and Predicts Neurological Events During Tuberculous Meningitis Treatment.

BACKGROUND: The Mycobacterium tuberculosis load in the brain of individuals with tuberculous meningitis (TBM) may reflect the host's ability to control the pathogen, determine disease severity, and determine treatment outcomes. METHODS: We used the GeneXpert assay to measure the pretreatment M. tuberculosis load in cerebrospinal fluid (CSF) specimens from 692 adults with TBM. We sought to understand the relationship between CSF bacterial load and inflammation, and their respective impact on disease severity and treatment outcomes. RESULTS: A 10-fold higher M. tuberculosis load was associated with increased disease severity (odds ratio, 1.59; P = .001 for the comparison between grade 1 and grade 3 severity), CSF neutrophil count (r = 0.364 and P < .0001), and cytokine concentrations (r = 0.438 and P < .0001). A high M. tuberculosis load predicted new neurological events after starting treatment (P = .005, by multinomial logistic regression) but not death. Patients who died had an attenuated inflammatory response at the start of treatment, with reduced cytokine concentrations as compared to survivors. In contrast, patients with high pretreatment CSF bacterial loads, cytokine concentrations, and neutrophil counts were more likely to subsequently experience neurological events. CONCLUSIONS: The pretreatment GeneXpert-determined M. tuberculosis load may be a useful predictor of neurological complications occurring during TBM treatment. Given the evidence for the divergent pathogenesis of TBM-associated neurological complications and deaths, therapeutic strategies to reduce them may need reassessment.

Relationship between Cell Receptors and Tumor Cell Sensitivity to Oncolytic Enteroviruses.

Replicative ability of 5 oncolytic enterovirus strains was evaluated on a panel of 18 human normal and tumor cells. The capacity of each cell line to support replication of enterovirus strains varied. Cell lines weakly replicating one virus could be highly sensitive to another viral strain. Differences in the expression of CXADR cell receptor did not correlate with susceptibility to infection and replication of Coxsackie B virus, but complete inactivation of CXADR gene and poliovirus receptor gene (PVR) led to loss of the sensitivity to Coxsackie B5 and poliovirus, respectively. Detection of additional expression markers will contribute to understanding the causes of different sensitivity of tumor cells to viruses.

Genetic association between leg conformation in young pigs and sow longevity.

Longevity is important in pig production with respect to both economic and ethical aspects. Direct selection for longevity might be ineffective because 'true' longevity can only be recorded when a sow has been culled or died. Thus, indirect selection for longevity using information from other traits that can be recorded early in life and are genetically correlated with longevity might be an alternative. Leg conformation has been included in many breeding schemes for a number of years. However, proving that leg conformation traits are good early indicators for longevity still remains. Our aim was to study genetic associations between leg conformation traits of young (5 months; 100 kg) Swedish Yorkshire pigs in nucleus herds and longevity traits of sows in nucleus and multiplier herds. Data included 97 533 animals with information on conformation (Movement and Overall score) recorded at performance testing and 26 962 sows with information on longevity. The longevity traits were as follows: stayability from 1st to 2nd parity, lifetime number of litters and lifetime number of born alive piglets. Genetic analyses were performed with both linear models using REML and linear-threshold models using Bayesian methods. Heritabilities estimated using the Bayesian method were higher than those estimated using REML, ranging from 0.10 to 0.24 and 0.07 to 0.20, respectively. All estimated genetic correlations between conformation and longevity traits were significant and favourable. Heritabilities and genetic correlations between conformation and longevity indicate that selection on leg conformation should improve sow longevity.

When private actors matter: Information-sharing network and surveillance of Highly Pathogenic Avian Influenza in Vietnam.

The effectiveness of animal health surveillance systems depends on their capacity to gather sanitary information from the animal production sector. In order to assess this capacity we analyzed the flow of sanitary information regarding Highly Pathogenic Avian Influenza (HPAI) suspicions in poultry in Vietnam. Participatory methods were applied to assess the type of actors and likelihood of information sharing between actors in case of HPAI suspicion in poultry. While the reporting of HPAI suspicions is mandatory, private actors had more access to information than public actors. Actors of the upstream sector (medicine and feed sellers) played a key role in the diffusion of information. The central role of these actors and the influence of the information flow on the adoption by poultry production stakeholders of behaviors limiting (e.g. prevention measures) or promoting disease transmission (e.g. increased animal movements) should be accounted for in the design of surveillance and control programs.

Comparison of experimental infection with northern and southern Vietnamese strains of highly pathogenic porcine reproductive and respiratory syndrome virus.

The aim of this study was to compare the virulence of northern and southern Vietnamese strains of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) as assessed by the level of viral replication, gross and microscopical lung lesions and virus distribution in experimentally infected pigs. The northern and southern Vietnamese HP-PRRSV strains share 96.7% (non-structural protein 2) and 99.3% (open reading frame 5) nucleotide identity. On experimental challenge, approximately 50% of pigs infected with northern Vietnamese HP-PRRSV died, while death was not observed in any pigs infected with southern Vietnamese HP-PRRSV. Mean viral titres (expressed as log(10)TCID(50)/ml) were significantly (P <0.05) higher in sera and lungs from pigs infected with the northern Vietnamese HP-PRRSV than from those infected with the southern Vietnamese strain at multiple time points. Lung lesion scores and PRRSV antigen within pulmonary and lymphoid lesions were significantly (P <0.05) higher in pigs infected with northern Vietnamese HP-PRRSV than in those receiving southern Vietnamese HP-PRRSV at multiple time points. PRRSV antigens were observed in cardiac myocytes, gastric and renal tubular epithelial cells and astrocytes and microglia of white matter in the brain from pigs infected with the northern Vietnamese HP-PRRSV strain only. Thus, genetic similarity did not predict the degree of virulence of these strains. Northern Vietnamese HP-PRRSV was more virulent and had extended tissue tropism when compared with southern Vietnamese HP-PRRSV.

Fourier transform infrared identification of the ν5(σu) fundamental of SiC5Si.

The Fourier transform infrared spectrum of SiC5Si was observed by trapping the vapor from the Nd:YAG laser ablation of sintered Si/C rods in solid Ar at ∼20 K. Measurements of (13)C and (29,30)Si isotopic shifts have enabled the identification of the ν5(σu) vibrational fundamental of the linear isomer of SiC5Si at 1590.8 ± 0.2 cm(-1). The results are in excellent agreement with the predictions of density functional theory calculations at the B3LYP/cc-pVDZ level. A second fundamental, ν4(σu), can only be tentatively identified at 2021.0 cm(-1), because its isotopic shifts are overlapped by absorptions from other species.

Detection of zeptomole quantities of nonfluorescent molecules in a 10(1) nm nanochannel by thermal lens microscopy.

Nanofluidics in 10(1) nm space, whose scale is comparable to the electric double layer (EDL) and the size of biomolecules, promises novel functional analytical devices. However, the detection, which is indispensable to the integrated chemical system, is still challenging in such an ultra-small space. Previously, we reported a differential interference contrast thermal lens microscope (DIC-TLM) based on the photothermal interferometry principle and succeeded in detection of nonfluorescent molecules in 10(2) nm spaces. However, the thermal diffusion into substrates becomes a problem for detection in 10(1) nm spaces. The DIC-TLM signals are significantly cancelled out in spaces much smaller than the confocal length (∼10(2) nm), which makes DIC-TLM detection in 10(1) nm space quite difficult. To overcome this problem, we propose a new channel structure that benefits the thermal diffusion and sensitivity enhancement in DIC-TLM by employing TiO2 as a substrate material for compensating the signal cancellation effect. As a result, DIC-TLM detection of nonfluorescent molecules (800 molecules) was successfully demonstrated in a nanochannel with a depth of 50 nm. The developed detection method will contribute to the functional nanofluidic devices utilizing 10(1) nm spaces.

Fourier transform infrared isotopic study of SiC5: identification of the ν(4)(σ) mode.

SiC5 in its (3)Σ ground state has been produced by trapping the products from the laser evaporation of a sintered silicon-carbon rod in solid Ar. For the first time a vibrational fundamental has been measured, the ν4(σ) asymmetric stretch at 936.9 ± 0.2 cm(-1). Comparison of observed (13)C and (29,30)Si isotopic shifts with the predictions of DFT-B3LYP/cc-pVDZ calculations confirms the identification.

Detection and molecular characterization of noroviruses and sapoviruses in children admitted to hospital with acute gastroenteritis in Vietnam.

Noroviruses (NoV) and sapoviruses (SaV) are recognized as important causes of acute gastroenteritis in children worldwide. In this study, the prevalence and genetic variability of NoV and SaV were determined in hospitalized children <5 years of age with acute gastroenteritis in Hanoi, Vietnam. A total of 501 fecal specimens collected between November-2007 and October-2008, that previously had been tested for rotavirus (RV), were tested for NoV and SaV by realtime RT-PCR. Positive samples were genotyped by conventional RT-PCR followed by sequencing. GII NoV was detected in 180 (36%) and SaV in 7 (1.4%) of the samples. NoV was detected year-round ranging from 9.5% in April to 81.5% in September among RV negative samples. NoV GII.4 Minerva (2006b) was the dominant genotype (93%) with a few other genotypes detected including GII.3 (4.4%), GII.13 (1.7%), and GII.2 (0.6%) but no GI strains. Only GI and GII SaV strains were detected in this study. No difference in NoV prevalence between age groups was noted. Frequency of vomiting or fever was similar between children with NoV and RV infection, yet, NoV caused diarrhea with longer duration. In conclusion, NoV is the second most frequent cause of diarrhea in hospitalized children in North Vietnam.

Bovine fasciolosis in the human fasciolosis hyperendemic Binh Dinh province in Central Vietnam.

A cross-sectional survey on bovine fasciolosis was conducted in the Binh Dinh province of Central Vietnam that was previously identified as hyperendemic for human fasciolosis. In Vietnam, both pure Fasciola gigantica and hybrid and/or introgressed populations of liverflukes bearing genetic material from both Fasciola hepatica and F. gigantica infect humans and animals. In this study, 825 cattle were randomly selected from 8 of the 11 provincial districts for faecal collection; blood samples were taken from 400 of these animals. Fasciola eggs and antibodies against Fasciola were detected by a quantitative sedimentation method and an Enzyme Linked Immunosorbent Assay, respectively. Overall, 54.9% of the animals were shedding Fasciola eggs while 72.2% were Fasciola seropositive. Animals under two years showed lower Fasciola infection rates than older animals. There were no differences in infection rates between districts. These results indicate a very high prevalence of Fasciola infections in cattle in Binh Dinh province. It is concluded that a fasciolosis control programme should be designed in this region aiming at reducing infection in both cattle and humans.

Assessment of a 27-kDa antigen in enzyme-linked immunosorbent assay for the diagnosis of fasciolosis in Vietnamese patients.

Fasciolosis has emerged as an important zoonotic disease in many parts of the world. In recent years, an increasing number of human cases were reported in Vietnam. In this study, the 27-kDa component protein from the excretory/secretory production of adult Fasciola gigantica, purified by high performance liquid chromatography, was assessed in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola spp. for diagnosis of human fasciolosis. The ELISA showed a high sensitivity (100%) and specificity (97.67%) when tested on patients with fasciolosis, other parasitic infections, cholangiocarcinoma and on healthy controls. The assay was applied for diagnosis on 143 patients in the Viet Duc-Hanoi hospital who presented with clinical signs of liver disease and lesions in their livers as shown by imaging techniques. Antibodies were found in 37 (25.9%) of these patients, of whom only 3 shed Fasciola eggs in their stools (2.1%). The excellent response to triclabendazole treatment of 37 sero-positive patients confirmed the diagnosis of fasciolosis. This study demonstrated the diagnostic potential for human fasciolosis of the 27-kDa antigen ELISA. Fasciolosis should be considered in the differential diagnosis of hepatic disease in Vietnam.