Biochemical Parameters for Longitudinal Monitoring of Liver Function in Rat Models of Partial Hepatectomy Following Liver Injury

Background

While evaluation of liver function in preclinical animal studies is commonly performed at selected time-points by invasive determination of the liver/body weight ratio and histological analyses, the validation of longitudinal measurement tools for monitoring liver function are of major interest.

Aims

To longitudinally evaluate serum cholinesterase (CHE) and total serum bilirubin (TSB) levels as non-invasive markers to determine injury- and partial hepatectomy (PHx)-induced alterations of liver function in rats.

Methods

Male and female Lewis rats were subjected to either methionine/choline deficient (MCD) diet or treatment with FOLFOX chemotherapy prior to PHx. Body weight and CHE/TSB levels are determined weekly. Following PHx and at the study end, histological analyses of liver tissue are performed.

Results

Following MCD diet, but not after FOLFOX chemotherapy treatment, results indicate gender-specific alterations in serum CHE levels and gender-independent alterations in TSB levels. Likewise, histological analyses of resected liver parts indicate significant liver injury following MCD-diet, but not following FOLFOX treatment. While TSB levels rapidly recover following MCD diet/FOLFOX treatment combined with a PHx, serum CHE levels are subject to significant model- and gender-specific differences, despite full histopathological recovery of liver tissue.

Conclusions

Longitudinal measurements of serum CHE levels and TSB levels in rats are highly complementary as non-invasive parameters for evaluation of liver injury and/or recovery.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Young Children's Probability of Dying Before and After Their Mother's Death: A Rural South African Population-Based Surveillance Study

Background

There is evidence that a young child''s risk of dying increases following the mother''s death, but little is known about the risk when the mother becomes very ill prior to her death. We hypothesized that children would be more likely to die during the period several months before their mother''s death, as well as for several months after her death. Therefore we investigated the relationship between young children''s likelihood of dying and the timing of their mother''s death and, in particular, the existence of a critical period of increased risk.

Methods and Findings

Data from a health and socio-demographic surveillance system in rural South Africa were collected on children 0–5 y of age from 1 January 1994 to 31 December 2008. Discrete time survival analysis was used to estimate children''s probability of dying before and after their mother''s death, accounting for moderators. 1,244 children (3% of sample) died from 1994 to 2008. The probability of child death began to rise 6–11 mo prior to the mother''s death and increased markedly during the 2 mo immediately before the month of her death (odds ratio [OR] 7.1 [95% CI 3.9–12.7]), in the month of her death (OR 12.6 [6.2–25.3]), and during the 2 mo following her death (OR 7.0 [3.2–15.6]). This increase in the probability of dying was more pronounced for children whose mothers died of AIDS or tuberculosis compared to other causes of death, but the pattern remained for causes unrelated to AIDS/tuberculosis. Infants aged 0–6 mo at the time of their mother''s death were nine times more likely to die than children aged 2–5 y. The limitations of the study included the lack of knowledge about precisely when a very ill mother will die, a lack of information about child nutrition and care, and the diagnosis of AIDS deaths by verbal autopsy rather than serostatus.

Conclusions

Young children in lower income settings are more likely to die not only after their mother''s death but also in the months before, when she is seriously ill. Interventions are urgently needed to support families both when the mother becomes very ill and after her death. Please see later in the article for the Editors'' Summary     

Bundles of Spider Silk,Braided into Sutures,Resist Basic Cyclic Tests: Potential Use for Flexor Tendon Repair

Repair success for injuries to the flexor tendon in the hand is often limited by the in vivo behaviour of the suture used for repair. Common problems associated with the choice of suture material include increased risk of infection, foreign body reactions, and inappropriate mechanical responses, particularly decreases in mechanical properties over time. Improved suture materials are therefore needed. As high-performance materials with excellent tensile strength, spider silk fibres are an extremely promising candidate for use in surgical sutures. However, the mechanical behaviour of sutures comprised of individual silk fibres braided together has not been thoroughly investigated. In the present study, we characterise the maximum tensile strength, stress, strain, elastic modulus, and fatigue response of silk sutures produced using different braiding methods to investigate the influence of braiding on the tensile properties of the sutures. The mechanical properties of conventional surgical sutures are also characterised to assess whether silk offers any advantages over conventional suture materials. The results demonstrate that braiding single spider silk fibres together produces strong sutures with excellent fatigue behaviour; the braided silk sutures exhibited tensile strengths comparable to those of conventional sutures and no loss of strength over 1000 fatigue cycles. In addition, the braiding technique had a significant influence on the tensile properties of the braided silk sutures. These results suggest that braided spider silk could be suitable for use as sutures in flexor tendon repair, providing similar tensile behaviour and improved fatigue properties compared with conventional suture materials.     

Increased Respiratory Disease Mortality at a Microwave Popcorn Production Facility with Worker Risk of Bronchiolitis Obliterans

Background

Bronchiolitis obliterans, an irreversible lung disease, was first associated with inhalation of butter flavorings (diacetyl) in workers at a microwave popcorn company. Excess rates of lung-function abnormalities were related to cumulative diacetyl exposure. Because information on potential excess mortality would support development of permissible exposure limits for diacetyl, we investigated respiratory-associated mortality during 2000–2011 among current and former workers at this company who had exposure to flavorings and participated in cross-sectional surveys conducted between 2000–2003.

Methods

We ascertained workers'' vital status through a Social Security Administration search. Causes of death were abstracted from death certificates. Because bronchiolitis obliterans is not coded in the International Classification of Disease 10^th revision (ICD-10), we identified respiratory mortality decedents with ICD-10 codes J40–J44 which encompass bronchitis (J40), simple and mucopurulent chronic bronchitis (J41), unspecified chronic bronchitis (J42), emphysema (J43), and other chronic obstructive pulmonary disease (COPD) (J44). We calculated expected number of deaths and standardized mortality ratios (SMRs) with 95% confidence intervals (CI) to determine if workers exposed to diacetyl experienced greater respiratory mortality than expected.

Results

We identified 15 deaths among 511 workers. Based on U.S. population estimates, 17.39 deaths were expected among these workers (SMR = 0.86; CI:0.48-1.42). Causes of death were available for 14 decedents. Four deaths among production and flavor mixing workers were documented to have a multiple cause of ‘other COPD’ (J44), while 0.98 ‘other COPD’-associated deaths were expected (SMR = 4.10; CI:1.12–10.49). Three of the 4 ‘other COPD’-associated deaths occurred among former workers and workers employed before the company implemented interventions reducing diacetyl exposure in 2001.

Conclusion

Workers at the microwave popcorn company experienced normal rates of all-cause mortality but higher rates of COPD-associated mortality, especially workers employed before the company reduced diacetyl exposure. The demonstrated excess in COPD-associated mortality suggests continued efforts to lower flavoring exposure are prudent.     

The Loss of Gnai2 and Gnai3 in B Cells Eliminates B Lymphocyte Compartments and Leads to a Hyper-IgM Like Syndrome

B lymphocytes are compartmentalized within lymphoid organs. The organization of these compartments depends upon signaling initiated by G-protein linked chemoattractant receptors. To address the importance of the G-proteins Gα_i2 and Gα_i3 in chemoattractant signaling we created mice lacking both proteins in their B lymphocytes. While bone marrow B cell development and egress is grossly intact; mucosal sites, splenic marginal zones, and lymph nodes essentially lack B cells. There is a partial block in splenic follicular B cell development and a 50-60% reduction in splenic B cells, yet normal numbers of splenic T cells. The absence of Gα_i2 and Gα_i3 in B cells profoundly disturbs the architecture of lymphoid organs with loss of B cell compartments in the spleen, thymus, lymph nodes, and gastrointestinal tract. This results in a severe disruption of B cell function and a hyper-IgM like syndrome. Beyond the pro-B cell stage, B cells are refractory to chemokine stimulation, and splenic B cells are poorly responsive to antigen receptor engagement. Gα_i2 and Gα_i3 are therefore critical for B cell chemoattractant receptor signaling and for normal B cell function. These mice provide a worst case scenario of the consequences of losing chemoattractant receptor signaling in B cells.     

Genotypic variation in the persistence of transgenerational responses to seasonal cues*

Phenotypes respond to environments experienced directly by an individual, via phenotypic plasticity, or to the environment experienced by ancestors, via transgenerational environmental effects. The adaptive value of environmental effects depends not only on the strength and direction of the induced response but also on how long the response persists within and across generations, and how stably it is expressed across environments that are encountered subsequently. Little is known about the genetic basis of those distinct components, or even whether they exhibit genetic variation. We tested for genetic differences in the inducibility, temporal persistence, and environmental stability of transgenerational environmental effects in Arabidopsis thaliana. Genetic variation existed in the inducibility of transgenerational effects on traits expressed across the life cycle. Surprisingly, the persistence of transgenerational effects into the third generation was uncorrelated with their induction in the second generation. Although environmental effects for some traits in some genotypes weakened over successive generations, others were stronger or even in the opposite direction in more distant generations. Therefore, transgenerational effects in more distant generations are not merely caused by the retention or dissipation of those expressed in prior generations, but they may be genetically independent traits with the potential to evolve independently.     

Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids,viral vectors and cell lines

As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems.