Adaptation and application of a two-plasmid inducible CRISPR-Cas9 system in Clostridium beijerinckii.

Recent developments in CRISPR technologies have opened new possibilities for improving genome editing tools dedicated to the Clostridium genus. In this study we adapted a two-plasmid tool based on this technology to enable scarless modification of the genome of two reference strains of Clostridium beijerinckii producing an Acetone/Butanol/Ethanol (ABE) or an Isopropanol/Butanol/Ethanol (IBE) mix of solvents. In the NCIMB 8052 ABE-producing strain, inactivation of the SpoIIE sporulation factor encoding gene resulted in sporulation-deficient mutants, and this phenotype was reverted by complementing the mutant strain with a functional spoIIE gene. Furthermore, the fungal cellulase-encoding celA gene was inserted into the C. beijerinckii NCIMB 8052 chromosome, resulting in mutants with endoglucanase activity. A similar two-plasmid approach was next used to edit the genome of the natural IBE-producing strain C. beijerinckii DSM 6423, which has never been genetically engineered before. Firstly, the catB gene conferring thiamphenicol resistance was deleted to make this strain compatible with our dual-plasmid editing system. As a proof of concept, our dual-plasmid system was then used in C. beijerinckii DSM 6423 ΔcatB to remove the endogenous pNF2 plasmid, which led to a sharp increase of transformation efficiencies.

Sporulation in solventogenic and acetogenic clostridia.

The Clostridium genus harbors compelling organisms for biotechnological production processes; while acetogenic clostridia can fix C1-compounds to produce acetate and ethanol, solventogenic clostridia can utilize a wide range of carbon sources to produce commercially valuable carboxylic acids, alcohols, and ketones by fermentation. Despite their potential, the conversion by these bacteria of carbohydrates or C1 compounds to alcohols is not cost-effective enough to result in economically viable processes. Engineering solventogenic clostridia by impairing sporulation is one of the investigated approaches to improve solvent productivity. Sporulation is a cell differentiation process triggered in bacteria in response to exposure to environmental stressors. The generated spores are metabolically inactive but resistant to harsh conditions (UV, chemicals, heat, oxygen). In Firmicutes, sporulation has been mainly studied in bacilli and pathogenic clostridia, and our knowledge of sporulation in solvent-producing or acetogenic clostridia is limited. Still, sporulation is an integral part of the cellular physiology of clostridia; thus, understanding the regulation of sporulation and its connection to solvent production may give clues to improve the performance of solventogenic clostridia. This review aims to provide an overview of the triggers, characteristics, and regulatory mechanism of sporulation in solventogenic clostridia. Those are further compared to the current knowledge on sporulation in the industrially relevant acetogenic clostridia. Finally, the potential applications of spores for process improvement are discussed.Key Points• The regulatory network governing sporulation initiation varies in solventogenic clostridia.• Media composition and cell density are the main triggers of sporulation.• Spores can be used to improve the fermentation process.

l-Rhamnose Metabolism in Clostridium beijerinckii Strain DSM 6423.

Macroalgae (or seaweeds) are considered potential biomass feedstocks for the production of renewable fuels and chemicals. Their sugar composition is different from that of lignocellulosic biomasses, and in green species, including Ulva lactuca, the major sugars are l-rhamnose and d-glucose. C. beijerinckii DSM 6423 utilized these sugars in a U. lactuca hydrolysate to produce acetic acid, butyric acid, isopropanol, butanol, and ethanol (IBE), and 1,2-propanediol. d-Glucose was almost completely consumed in diluted hydrolysates, while l-rhamnose or d-xylose was only partially utilized. In this study, the metabolism of l-rhamnose by C. beijerinckii DSM 6423 was investigated to improve its utilization from natural resources. Fermentations on d-glucose, l-rhamnose, and a mixture of d-glucose and l-rhamnose were performed. On l-rhamnose, the cultures showed low growth and sugar consumption and produced 1,2-propanediol, propionic acid, and n-propanol in addition to acetic and butyric acids, whereas on d-glucose, IBE was the major product. On a d-glucose-l-rhamnose mixture, both sugars were converted simultaneously and l-rhamnose consumption was higher, leading to high levels of 1,2-propanediol (78.4 mM), in addition to 59.4 mM butanol and 31.9 mM isopropanol. Genome and transcriptomics analysis of d-glucose- and l-rhamnose-grown cells revealed the presence and transcription of genes involved in l-rhamnose utilization and in bacterial microcompartment (BMC) formation. These data provide useful insights into the metabolic pathways involved in l-rhamnose utilization and the effects on the general metabolism (glycolysis, early sporulation, and stress response) induced by growth on l-rhamnose.IMPORTANCE A prerequisite for a successful biobased economy is the efficient conversion of biomass resources into useful products, such as biofuels and bulk and specialty chemicals. In contrast to other industrial microorganisms, natural solvent-producing clostridia utilize a wide range of sugars, including C5, C6, and deoxy-sugars, for production of long-chain alcohols (butanol and 2,3-butanediol), isopropanol, acetone, n-propanol, and organic acids. Butanol production by clostridia from first-generation sugars is already a commercial process, but for the expansion and diversification of the acetone, butanol, and ethanol (ABE)/IBE process to other substrates, more knowledge is needed on the regulation and physiology of fermentation of sugar mixtures. Green macroalgae, produced in aquaculture systems, harvested from the sea or from tides, can be processed into hydrolysates containing mixtures of d-glucose and l-rhamnose, which can be fermented. The knowledge generated in this study will contribute to the development of more efficient processes for macroalga fermentation and of mixed-sugar fermentation in general.

[Accuracy and diagnostic utility of IgM in Bartonella henselae infections]. / Exactitud y utilidad diagnóstica de la IgM en infecciones por Bartonella henselae.

INTRODUCTION: Laboratory diagnosis of cat scratch disease (CSD) is based on the determination of specific antibodies anti-Bartonella henselae by different techniques. The CDC recommends IgG by immunofluorescent assay (IFA) as the gold standard. OBJECTIVE: To determine the accuracy and diagnostic utility of anti-B.henselae IgM by IFA for CSD. MATERIAL AND METHODS: Anti-B. henselae IgG was determined in serum of 108 patients with CSD suspicion; in addition, specific IgM was determined separately and blindly by two thoroughly trained laboratory professionals. We calculated sensitivity (S), specificity (Sp), predictive values both positive (PPV) and negative (NPV), and likelihood ratio (LR) for IgM positive (LR +) and negative (LR-). RESULTS: In 37 patients with positive anti-B.henselae IgG, IgM was positive in 16 and negative in 21; in 71 patients with negative IgG, IgM was negative in 69 and positive in 2. Therefore, IgM showed S 43%, E 97%, PPV 88%, NPV 77%, LR (+) 15 and LR (-) 0.58. CONCLUSIONS: The results show that a positive IgM supports, but a negative one does not rule out a B. henselae infection. Therefore, IgG should be still considered as the gold standard for the diagnosis of CSD.

Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a.

Clostridium species are re-emerging as biotechnological workhorses for industrial acetone-butanol-ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25-100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.

Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production.

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.

[Guideline for interpretation and report of the antibody to hepatitis C virus. Grupo de Desarrollo de la Guía ]. / Guía de interpretación y reporte del anticuerpo a hepatitis C.

Patients with hepatitis C virus (HCV) infection are detected by testing for the presence of antibodies to HCV (Anti-HCV). A positive Anti-HCV test represents a true positive result only in a variable proportion of subjects (35 to 95%). The qualitative interpretation as positive or negative Anti-HCV report is associated with a general lack of understanding regarding the interpretation of results, when more specific testing should be performed, and which tests should be considered for this purpose. Therefore, a substantial variation in supplemental testing practices exists among laboratories and physicians. This guideline was developed on the basis of the best available evidence to classify positive antibody in two (low and high) or three levels (very low, low and high) according to the signal to cutoff (S/CO) ratio: the very low level of the Anti-HCV identifies false-positive results and further diagnostic testing is not necessary. The low antibody level is frequently related with false-positive results and testing with Immunoblot is recommended; only Immunoblot-positive subjects require HCV RNA testing because of a low possibility of being viremic. The high Anti-HCV level is an accurate serological marker for predicting viremia and denotes the need of routine HCV RNA testing in order to efficiently confirm hepatitis C. Cost-effectiveness analysis, based on the Anti-HCV level, recommends the use of the two or three-levels to choose the confirmatory test of positive antibody. This approach can be implemented without increasing test costs because the S/CO ratio is automatically generated in most laboratory analyzers and would provide health care professionals with useful information for counseling and evaluating patients, to eliminate unwarranted notifications in cases of false antibody reactivity, and correctly identifying those Anti-HCV-positive patients who are infected and need antiviral treatment. The written report should include the antibody level (S/CO ratio), the type of the immunoassay applied and interpretation guideline. Anti-HCV testing is performed in multiple settings including blood banks or health department facilities; adoption of this Guideline for interpretation and report of the antibody to hepatitis C virus by laboratories and its implementation by clinicians will improve the accuracy for interpreting antibody result to determine the next step on hepatitis C diagnosis.

Model-driven approach for the production of butyrate from CO2/H2 by a novel co-culture of C. autoethanogenum and C. beijerinckii.

One-carbon (C1) compounds are promising feedstocks for the sustainable production of commodity chemicals. CO2 is a particularly advantageous C1-feedstock since it is an unwanted industrial off-gas that can be converted into valuable products while reducing its atmospheric levels. Acetogens are microorganisms that can grow on CO2/H2 gas mixtures and syngas converting these substrates into ethanol and acetate. Co-cultivation of acetogens with other microbial species that can further process such products, can expand the variety of products to, for example, medium chain fatty acids (MCFA) and longer chain alcohols. Solventogens are microorganisms known to produce MCFA and alcohols via the acetone-butanol-ethanol (ABE) fermentation in which acetate is a key metabolite. Thus, co-cultivation of an acetogen and a solventogen in a consortium provides a potential platform to produce valuable chemicals from CO2. In this study, metabolic modeling was implemented to design a new co-culture of an acetogen and a solventogen to produce butyrate from CO2/H2 mixtures. The model-driven approach suggested the ability of the studied solventogenic species to grow on lactate/glycerol with acetate as co-substrate. This ability was confirmed experimentally by cultivation of Clostridium beijerinckii on these substrates in batch serum bottles and subsequently in pH-controlled bioreactors. Community modeling also suggested that a novel microbial consortium consisting of the acetogen Clostridium autoethanogenum, and the solventogen C. beijerinckii would be feasible and stable. On the basis of this prediction, a co-culture was experimentally established. C. autoethanogenum grew on CO2/H2 producing acetate and traces of ethanol. Acetate was in turn, consumed by C. beijerinckii together with lactate, producing butyrate. These results show that community modeling of metabolism is a valuable tool to guide the design of microbial consortia for the tailored production of chemicals from renewable resources.

D-2,3-butanediol production due to heterologous expression of an acetoin reductase in Clostridium acetobutylicum.

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions, Clostridium acetobutylicum ATCC 824 (and strains derived from it) generates both d- and l-stereoisomers of acetoin, but because of the absence of an ACR enzyme, it does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under the control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89 to 90% of the natively produced acetoin was converted to 20 to 22 mM d-2,3-butanediol. The addition of a racemic mixture of acetoin led to the production of both d-2,3-butanediol and meso-2,3-butanediol. A metabolic network that is in agreement with the experimental data is proposed. Native 2,3-butanediol production is a first step toward a potential homofermentative 2-butanol-producing strain of C. acetobutylicum.

[Safe blood in the absence of viral infections due to HBV, HCV and HIV in serological window period in donors]. / Sangre segura en ausencia de infecciones virales por VHB, VHC y VIH en período de ventana serológica de donadores.

OBJECTIVE: To determine the prevalence of viral infections (HBV, HCV and HIV) in serological window period in blood donors screened with nucleic acid testing (NAT). MATERIALS AND METHODS: We assessed all blood donors from July 2008 to June 2009 at the Central Blood Bank of the Mexican Institute of Social Security. Medical history was made and provided an information brochure and self-exclusion questionnaire. All blood donors were tested with serological tests (Ag-HBVs, Anti-HCV and Anti-HIV) and molecular testing with NAT for HBV, HCV and HIV. The window period was defined with the positive NAT and negative serological test. RESULTS: During one year, we evaluated 47 847 blood donors. None subject was identified with viral infection (HBV, HCV and HIV) in serological window period. Positive serological testing were found for HBV in 78 (0.2%), 318 (0.7%) for HCV and 155 (0.3%) for HIV. Positive NAT was demonstrated only in donors with positive serology: 26 of 78 with HBV, 56 of 318 with HCV and 16 of 155 with HIV. CONCLUSION: This is the first study in México showed no viral infections (HBV, HCV and HIV) during serological window period in blood donors; The medical history and the self-exclusion questionnaire help to improve blood transfusion safety.

[Nosocomial transmission of hepatitis C associated with anesthesia procedures: a case-control study]. / Transmisión nosocomial de la hepatitis C asociada a procedimientos anestésicos: un estudio de casos y controles.

OBJECTIVE: Nosocomial transmission of hepatitis C virus (HCV) infection had been related with anesthesia procedures. The study aim was to measure the association between anesthesia procedures in cases with previous surgery and HCV infection. MATERIAL AND METHODS: In a case-control study were included subjects that attended to the Central Blood Bank of the West Medical National Center, Mexican Institute of the Social Security in Guadalajara, Jalisco between july 2005 and september 2007. Cases were patients with positive hepatitis C antibody (anti-HCV) confirmed by recombinant immunoblot assay (RIBA) and/or nucleic acid test (HCV RNA); the control group was blood donors with negative antibody. An exhaustive questionnaire about risk factors for hepatitis C, was applied. The risk of HCV infection was determined with the Odds Ratio (OR) and multivariate analysis was made by logistic regression. RESULTS: We included 362 subjects, 211 cases and 151 controls; in 70 (33.2%) cases were found significant association between the anesthesia procedures and HCV infection in patients with previous surgery (OR adjusted 2.44, CI 95% 1.44 - 4.11) CONCLUSION: This is the first study in México that demonstrate association between history of anesthesia procedures and HCV infection in cases with previous surgery.

Chemical Genetics Applied to Elucidate the Physiological Role of Stress-Signaling Molecules on the Wound-Induced Accumulation of Glucosinolates in Broccoli.

Wounding stress is an effective strategy to induce glucosinolate (GS) biosynthesis in broccoli. However, there is insufficient knowledge on the physiological and molecular mechanisms underlying this stress response. Herein, a chemical-genetic approach was applied to elucidate the role of jasmonic acid (JA), ethylene (ET), and reactive oxygen species (ROS) on the wound-induced biosynthesis of GS. Broccoli was processed into chops to induce wounding stress. Broccoli chops were treated with phenidone (PHEN) and diphenyleneiodonium chloride (DPI) as inhibitors of JA and ROS biosynthesis, respectively, whereas 1-methylcyclopropene (1-MCP) was applied as an inhibitor of ET action. Wounding stress induced the expression of genes related to the biosynthesis of indolic and aliphatic GS, which was correlated with the accumulation of GS and modulated by the inhibitors of signaling molecules applied. Results of gene expression analysis indicated that JA played a key role in the activation of most genes, followed by ROS. Furthermore, except for the CYP79B2 gene, PHEN and 1-MCP synergistically downregulated the expression of GS biosynthetic genes evaluated, showing that the interaction between JA and ET was fundamental to modulate GS biosynthesis. Results presented herein increased our knowledge of the physiological and molecular mechanisms governing the wound-induced biosynthesis of GS in broccoli.

High antibody level: an accurate serologic marker of viremia in asymptomatic people with hepatitis C infection.

BACKGROUND: The screening and diagnosis of hepatitis C virus (HCV) infection is initiated by testing for antibody to HCV (anti-HCV). A positive anti-HCV test in blood donors represents ongoing infection in only a variable proportion of individuals. Because a high anti-HCV level has been associated with viremia, a study was conducted to determine whether a high antibody level is an accurate serologic marker for viremia in asymptomatic anti-HCV-positive persons. STUDY DESIGN AND METHODS: In a diagnostic test study, we included 856 anti-HCV-positive blood donors in a blood bank at Guadalajara, Jalisco, Mexico, between 2002 and 2007. A third-generation amplified chemiluminescence assay (ChLIA HCV) was used to detect anti-HCV. A positive result of the qualitative nucleic acid test (HCV RNA) was considered the gold standard for viremia. RESULTS: By receiver operating characteristic analysis, the signal-to-cutoff (S/CO) ratio of 20 or more was chosen as optimal to identify viremia and so was defined as high anti-HCV level. There was a significant difference in the proportion of viremia between subjects with high antibody level and those with lower levels (93.7% vs. 1.8%, respectively; p < 0.001). A high antibody level showed a sensitivity for viremia of 96.6% (95% confidence interval [CI], 93.8%-98.1%), a specificity of 96.6% (95% CI, 94.8%-97.8%), and a likelihood ratio of 28.6 (95% CI, 18.4%-44.6%). CONCLUSION: A high antibody level (S/CO ratio >/=20 by ChLIA HCV) clearly divides the viremic from the nonviremic blood donors and functions as an accurate serologic marker to guide the use of routine HCV RNA testing to confirm hepatitis C infection.

Transcriptomic and Phenotypic Analysis of a spoIIE Mutant in Clostridium beijerinckii.

SpoIIE is a phosphatase involved in the activation of the first sigma factor of the forespore, σ F , during sporulation. A ΔspoIIE mutant of Clostridium beijerinckii NCIMB 8052, previously generated by CRISPR-Cas9, did not sporulate but still produced granulose and solvents. Microscopy analysis also showed that the cells of the ΔspoIIE mutant are elongated with the presence of multiple septa. This observation suggests that in C. beijerinckii, SpoIIE is necessary for the completion of the sporulation process, as seen in Bacillus and Clostridium acetobutylicum. Moreover, when grown in reactors, the spoIIE mutant produced higher levels of solvents than the wild type strain. The impact of the spoIIE inactivation on gene transcription was assessed by comparative transcriptome analysis at three time points (4 h, 11 h and 23 h). Approximately 5% of the genes were differentially expressed in the mutant compared to the wild type strain at all time points. Out of those only 12% were known sporulation genes. As expected, the genes belonging to the regulon of the sporulation specific transcription factors (σ F , σ E , σ G , σ K ) were strongly down-regulated in the mutant. Inactivation of spoIIE also caused differential expression of genes involved in various cell processes at each time point. Moreover, at 23 h, genes involved in butanol formation and tolerance, as well as in cell motility, were up-regulated in the mutant. In contrast, several genes involved in cell wall composition, oxidative stress and amino acid transport were down-regulated. These results indicate an intricate interdependence of sporulation and stationary phase cellular events in C. beijerinckii.

Very low hepatitis C antibody levels predict false-positive results and avoid supplemental testing.

BACKGROUND: False-positive results for hepatitis C virus antibody (anti-HCV) occur with unacceptable frequency in low-prevalence populations. The purpose of the study was to determine whether signal-to-cutoff (S/CO) ratios of anti-HCV assay-reactive samples could be used to discriminate false-positive from true-positive anti-HCV results and avoid the need for supplemental testing. STUDY DESIGN AND METHODS: Using receiver-operating characteristic curve, the cutoff point that identifies the major proportion (>/=95%) of false-positive results, with a minor proportion (<5%) of true-positive anti-HCV results, was determined. An anti-HCV assay (VITROS, Ortho Clinical Diagnostics) was used to detect the antibodies. The third-generation recombinant immunoblot assay and HCV RNA tests were performed on all included donors. Third-generation RIBA is the gold standard for identifying false-positive antibody results. RESULTS: A total of 649 anti-HCV-positive blood donors were identified. A S/CO ratio of less than 4.5, defining very low levels in this value, was the optimal cutoff point to identify false-positive results; 315 of 322 samples with very low levels were false-positive anti-HCV results (97.8%; 95% confidence interval [CI], 95.8%-99.0%) and 7 were true-positive (2.2%; 95% CI, 1.0%-4.3%). Viremia was detected in none of them. A direct relationship was observed between positive supplemental testing and increased antibody levels in the other 327 samples. CONCLUSION: The high prediction rate of false-positive anti-HCV results using very low levels by the Ortho VITROS anti-HCV assay safely avoids the need for supplemental testing.

[Parotiditis in Chile: clinical and molecular characterization of two cases in a highly vaccinated population]. / Parotiditis en Chile: caracterización clínica y molecular de dos casos en una población altamente inmunizada.

Mumps virus usually produces a benign infection characterized by increased parotid volume which, prior to vaccination, mainly affected children and adolescents. After the introduction of measles, mumps and rubella (MMR) vaccine, mumps incidence decreased dramatically. This intervention also produced a change in its clinical presentation, moving to young adult patients, with an increased risk of complications. We report two clinical mumps cases in young adults with different clinical presentations. In both cases, serologic assays were assessed and, in one case, a polymerase chain reaction (PCR) was performed in order to confirm the diagnosis. The isolated virus was characterized and identifed as G genotype, the same genotype observed during outbreaks in United States and Europe, and different to the vaccinal strain. Mumps virus is currently circulating in Chile and it is important to be aware of possible outbreaks. Viral diagnosis can be difficult, particularly in populations with high vaccination coverage. Therefore, the access to etiologic study through PCR and serology becomes more relevant in order to optimize clinical management and secondary prevention measures.

Evaluation of monoclonal antibodies that detect conserved proteins from Respiratory Syncytial Virus, Metapneumovirus and Adenovirus in human samples.

Human Respiratory Syncytial Virus (hRSV), human Metapneumovirus (hMPV) and Adenovirus (ADV), are three of the most prevalent viruses responsible for pneumonia and bronchiolitis in children and elderly worldwide, accounting for a high number of hospitalizations annually. Diagnosis of these viruses is required to take clinical actions that allow an appropriate patient management. Thereby, new strategies to design fast diagnostic methods are highly required. In the present work, six monoclonal antibodies (mAbs, two for each virus) specific for conserved proteins from hRSV, hMPV and ADV were generated and evaluated through different immunological techniques, based on detection of purified protein, viral particles and human samples. In vitro evaluation of these antibodies showed higher specificity and sensitivity than commercial antibodies tested in this study. These antibodies were used to design a sandwich ELISA tests that allowed the detection of hRSV, hMPV, and ADV in human nasopharyngeal swabs. We observed that hRSV and ADV were detected with sensitivity and specificity equivalent to a current Direct Fluorescence Assay (DFA) methodology. However, hMPV was detected with more sensitivity than DFA. Our data suggest that these new mAbs can efficiently identify infected samples and discriminate from patients infected with other respiratory pathogens.

Drug-related hepatotoxicity in a renal transplant recipient with long-term survival and hepatitis C.

Drug-related hepatotoxicity is more common in renal transplant (RT) recipients with chronic liver disease because drug metabolism is not as efficient in these individuals. We describe a long-term survivor (30 years) of renal transplantation with hepatitis C virus (HCV) and drug-related hepatotoxicity. Our patient, a 26-year-old male, developed uremic syndrome in May 1976 and received a renal allograft from a related, living donor with an identical human leukocyte antigen genotype in August 1976. Maintenance immunosuppression treatment consisted of azathioprine (AZA) and prednisone. In 1993, the patient tested negative for HCV antibody v1.0 (anti-HCV). In 2000, the patient had elevated aminotransferases, which was attributed to pravastatin treatment. Remission of this abnormality was achieved once pravastatin was discontinued. In 2003, the patient again exhibited elevated levels of aminotransferases and AZA-related hepatotoxicity was suspected; therefore, AZA was discontinued and treatment with mycophenolate mofetil was initiated, which led to normal aminotransferase levels. The patient tested positive for anti-HCV v3.0 and HCV RNA and a liver biopsy showed chronic hepatitis with moderate activity. Currently, the patient's renal transplant and liver are functional. In conclusion, hepatotoxic drugs should be used with caution in renal transplant recipients and close monitoring of liver function in patients with chronic viral hepatitis is crucial.

End-stage renal disease and hepatitis C infection: comparison of alanine aminotransferase levels and liver histology in patients with and without renal damage.

BACKGROUND AND AIM: To what extent the serum levels of alanine aminotransferase (ALT) are related to histological characteristics of liver damage caused by hepatitis C virus (HCV) infection among patients with end-stage renal disease (ESRD) remains unclear. METHODS: Patients with a positive anti-HCV antibody titer confirmed by supplemental tests were evaluated by liver biopsy. We compared ALT levels in patients with and without renal damage, with similar histological grades and stages of inflammation and fibrosis. Results: Patients were divided into two groups: patients with ESRD (n = 25) and patients without ESRD renal damage (n = 39). RESULTS: The ALT level was 42.1 +/- 24.3 IU/L for the ESRD group, compared with 109.9 +/- 55.8 IU/L for the non-ESRD group (P < 0.001). Liver inflammation (modified Knodell grade) was 4.0 +/- 2.1 in the ESRD group versus 5.2 +/- 2.4 in the non- ESRD group; fibrosis (6-point scale) was 1.1 +/- 1.2 versus 1.7 +/- 1.5, respectively. CONCLUSIONS: Despite histological evidence of liver inflammation, ALT levels in the ESRD group were normal, while ALT levels were significantly higher in the non-ESRD group with similar levels of liver inflammation. In conclusion, ALT levels are not a useful indicator of HCV infection in patients with ESRD and liver biopsies should be recommended for kidney transplant candidates.

A two-plasmid inducible CRISPR/Cas9 genome editing tool for Clostridium acetobutylicum.

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.