Evaluation of a novel ELISA test using synthetic mycolic acid antigens for serodiagnosis of non-tuberculous mycobacterial (NTM) infections.

The diagnosis of non-tuberculous mycobacteria (NTM) is a particular challenge in people with cystic fibrosis. Current standard diagnostic approaches rely on serial sputum culture, which is resource demanding, dependent on patient expectoration and may be compromised by excessive decontamination, conventional bacterial overgrowth and masking by concomitant oral and nebulised antibiotics. An alternative rapid, reliable and inexpensive diagnostic method is therefore urgently needed. Serum of patients with Mycobacterium abscessus infection and chronic suppurative lung disease without NTM infection was tested against an array of novel synthetic mycolic acids, identical or similar to natural components of mycobacterial cell walls, and glycopeptidolipid (GPL)-core antigen, which has previously been investigated in Mycobacterium avium pulmonary infection. Diagnostic accuracy of individual antigens and combination of various antigens were calculated. An ELISA using individual trehalose dimycolates and GPL-core antigen was able to effectively distinguish serum from infected and non-infected individuals with a specificity of 88% and a sensitivity of up to 88%, which increased to 88% sensitivity and 93% specificity by combining several antigens in the test. These results suggest synthetic mycolic acid antigens, used individually or in combination with GPL-core antigen could be successfully used to distinguish patients with M. abscessus infection from disease controls.

National TB program shortages as potential factor for poor-quality TB care cascade: Healthcare workers' perspective from Beira, Mozambique.

BACKGROUND: Mozambique is one of the countries with the deadly implementation gaps in the tuberculosis (TB) care and services delivery. In-hospital delays in TB diagnosis and treatment, transmission and mortality still persist, in part, due to poor-quality of TB care cascade. OBJECTIVE: We aimed to assess, from the healthcare workers' (HCW) perspective, factors associated with poor-quality TB care cascade and explore local sustainable suggestions to improve in-hospital TB management. METHODS: In-depth interviews and focus group discussions were conducted with different categories of HCW. Audio-recording and written notes were taken, and content analysis was performed through atlas.ti7. RESULTS: Bottlenecks within hospital TB care cascade, lack of TB staff and task shifting, centralized and limited time of TB laboratory services, and fear of healthcare workers getting infected by TB were mentioned to be the main factors associated with implementation gaps. Interviewees believe that task shifting from nurses to hospital auxiliary workers, and from higher and well-trained to lower HCW are accepted and feasible. The expansion and use of molecular TB diagnostic tools are seen by the interviewees as a proper way to fight effectively against both sensitive and MDR TB. Ensuring provision of N95 respiratory masks is believed to be an essential requirement for effective engagement of the HCW on high-quality in-hospital TB care. For monitoring and evaluation, TB quality improvement teams in each health facility are considered to be an added value. CONCLUSION: Shortage of resources within the national TB control programme is one of the potential factors for poor-quality of the TB care cascade. Task shifting of TB care and services delivery, decentralization of the molecular TB diagnostic tools, and regular provision of N95 respiratory masks should contribute not just to reduce the impact of resource scarceness, but also to ensure proper TB diagnosis and treatment to both sensitive and MDR TB.

Community-wide Screening for Tuberculosis in a High-Prevalence Setting.

BACKGROUND: The World Health Organization has set ambitious targets for the global elimination of tuberculosis. However, these targets will not be achieved at the current rate of progress. METHODS: We performed a cluster-randomized, controlled trial in Ca Mau Province, Vietnam, to evaluate the effectiveness of active community-wide screening, as compared with standard passive case detection alone, for reducing the prevalence of tuberculosis. Persons 15 years of age or older who resided in 60 intervention clusters (subcommunes) were screened for pulmonary tuberculosis, regardless of symptoms, annually for 3 years, beginning in 2014, by means of rapid nucleic acid amplification testing of spontaneously expectorated sputum samples. Active screening was not performed in the 60 control clusters in the first 3 years. The primary outcome, measured in the fourth year, was the prevalence of microbiologically confirmed pulmonary tuberculosis among persons 15 years of age or older. The secondary outcome was the prevalence of tuberculosis infection, as assessed by an interferon gamma release assay in the fourth year, among children born in 2012. RESULTS: In the fourth-year prevalence survey, we tested 42,150 participants in the intervention group and 41,680 participants in the control group. A total of 53 participants in the intervention group (126 per 100,000 population) and 94 participants in the control group (226 per 100,000) had pulmonary tuberculosis, as confirmed by a positive nucleic acid amplification test for Mycobacterium tuberculosis (prevalence ratio, 0.56; 95% confidence interval [CI], 0.40 to 0.78; P<0.001). The prevalence of tuberculosis infection in children born in 2012 was 3.3% in the intervention group and 2.6% in the control group (prevalence ratio, 1.29; 95% CI, 0.70 to 2.36; P = 0.42). CONCLUSIONS: Three years of community-wide screening in persons 15 years of age or older who resided in Ca Mau Province, Vietnam, resulted in a lower prevalence of pulmonary tuberculosis in the fourth year than standard passive case detection alone. (Funded by the Australian National Health and Medical Research Council; ACT3 Australian New Zealand Clinical Trials Registry number, ACTRN12614000372684.).

Climate control on banded iron formations linked to orbital eccentricity.

Astronomical forcing associated with Earth's orbital and inclination parameters ("Milankovitch" forcing) exerts a major control on climate as recorded in the sedimentary rock record, but its influence in deep time is largely unknown. Banded iron formations, iron-rich marine sediments older than 1.8 billion years, offer unique insight into the early Earth's environment. Their origin and distinctive layering have been explained by various mechanisms, including hydrothermal plume activity, the redox evolution of the oceans, microbial and diagenetic processes, sea level fluctuations, and seasonal or tidal forcing. However, their potential link to past climate oscillations remains unexplored. Here we use cyclostratigraphic analysis combined with high-precision uranium-lead dating to investigate the potential influence of Milankovitch forcing on their deposition. Field exposures of the 2.48-billion-year-old Kuruman Banded Iron Formation reveal a well-defined hierarchical cycle pattern in weathering profile that is laterally continuous over at least 250 kilometres. The isotopic ages constrain the sedimentation rate at 10 m/Myr and link the observed cycles to known eccentricity oscillations with periods of 405 thousand and about 1.4 to 1.6 million years. We conclude that long-period, Milankovitch-forced climate cycles exerted a primary control on large-scale compositional variations in banded iron formations.

Eliminating latent tuberculosis in low-burden settings: are the principal beneficiaries to be disadvantaged groups or the broader population?

Tuberculosis (TB) remains a leading cause of morbidity and mortality worldwide, and the burdens of this disease continue to track prior disadvantage. In order to galvanise a coordinated global response, WHO has recently launched the End TB Campaign that aims to eliminate TB by 2050. Key to this is the introduction of population screening programmes in low-burden settings to identify and treat people who have latent TB infection (LTBI). The defining features of LTBI are: that it is not an active disease but confers an increased risk of disease; the socially disadvantaged are those most in danger and uncertainty persists as to who will be harmed or benefitted from screening-led prophylactic interventions. Systematic screening programmes that include surveillance, case-finding and treatment of asymptomatic individuals inevitably redistribute the risk of harms and the potential for benefits within a population. The extent to which those targeted within such programmes should be exposed to higher levels of risk in the pursuit of individual or community benefits requires careful consideration prior to implementation. As currently construed, it remains unclear who stands to benefit most from how LTBI screening in high-income countries is being organised, and whose health is being prioritised: members of disadvantaged groups or the broader community. Unless the aims of LTBI screening programmes in these settings are made transparent and their prioritisation ethically justified, there is a significant danger that such a targeted intervention will further disadvantage those who have the least capacity to bear the burdens of TB elimination.

DiPOLE: a 10 J, 10 Hz cryogenic gas cooled multi-slab nanosecond Yb:YAG laser.

The Diode Pumped Optical Laser for Experiments (DiPOLE) project at the Central Laser Facility aims to develop a scalable, efficient high pulse energy diode pumped laser amplifier system based on cryogenic gas cooled, multi-slab ceramic Yb:YAG technology. We present recent results obtained from a scaled down prototype laser system designed for operation at 10 Hz pulse repetition rate. At 140 K, the system generated 10.8 J of energy in a 10 ns pulse at 1029.5 nm when pumped by 48 J of diode energy at 940 nm, corresponding to an optical to optical conversion efficiency of 22.5%. To our knowledge, this represents the highest pulse energy obtained from a cryo cooled Yb laser to date and the highest efficiency achieved by a multi-Joule diode pumped solid state laser system. Additionally, we demonstrated shot-to-shot energy stability of 0.85% rms for the system operated at 7 J, 10 Hz during several runs lasting up to 6 hours, with more than 50 hours in total. We also demonstrated pulse shaping capability and report on beam, wavefront and focal spot quality.

High-efficiency 10 J diode pumped cryogenic gas cooled Yb:YAG multislab amplifier.

We report on the first demonstration of a diode-pumped, gas cooled, cryogenic multislab Yb:YAG amplifier. The performance was characterized over a temperature range from 88 to 175 K. A maximum small-signal single-pass longitudinal gain of 11.0 was measured at 88 K. When amplifying nanosecond pulses, recorded output energies were 10.1 J at 1 Hz in a four-pass extraction geometry and 6.4 J at 10 Hz in a three-pass setup, corresponding to optical to optical conversion efficiencies of 21% and 16%, respectively. To our knowledge, this represents the highest pulse energy so far obtained from a cryo-cooled Yb-laser and the highest efficiency from a multijoule diode pumped solid-state laser system.

SCD1 inhibition causes cancer cell death by depleting mono-unsaturated fatty acids.

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence.

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21(/Cip) and p27(/Kip1). Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.

A number of proteins and drugs have been implicated in the process of transcriptional elongation by RNA polymerase (Pol) II, but the factors that govern the elongation rate (nucleotide additions per min) and processivity (nucleotide additions per initiation event) in vivo are poorly understood. Here, we show that a mutation in the Rpb2 subunit of Pol II reduces both the elongation rate and processivity in vivo. In contrast, none of the putative elongation factors tested affect the elongation rate, although mutations in the THO complex and in Spt4 significantly reduce processivity. The drugs 6-azauracil and mycophenolic acid reduce both the elongation rate and processivity, and this processivity defect is aggravated by mutations in Spt4, TFIIS, and CTDK-1. Our results suggest that, in vivo, a reduced rate of Pol II elongation leads to premature dissociation along the chromatin template and that Pol II processivity can be uncoupled from elongation rate.

Effect of bacterial endotoxin and middle ear effusion on ciliary activity: implications for otitis media.

OBJECTIVES/HYPOTHESIS: Otitis media with effusion (OME) is the most common cause of childhood deafness. The pathogenesis is not fully understood, especially the reasons for failure of mucociliary clearance of the middle ear. It is not clear whether the cilia function normally in the middle ear and eustachian tube in the chronic phase of otitis media with effusion. However, impaired ciliary function in primary ciliary dyskinesia is known to be frequently associated with the development of otitis media with effusion. We hypothesized that endotoxin or the bacterial products in middle ear fluid in otitis media with effusion would adversely affect ciliary activity, thereby contributing to the pathogenesis of the disease. STUDY DESIGN: Laboratory-based study of human ciliary activity with reference to otitis media with effusion. METHODS: We have studied the activity of human adenoidal cilia under various conditions. Ciliary activity in the presence of Haemophilus influenzae endotoxin additions (at varying concentrations) to cultured adenoidal explants has been measured. In addition, ciliary activity of these explants was also observed after addition of middle ear effusion aspirated from patients. RESULTS: We have shown that endotoxin in concentrations far in excess of those found in the middle ear with chronic otitis media with effusion had no effect on ciliary activity. Furthermore, ciliary activity was completely unaffected by the presence of middle ear effusion. CONCLUSION: There is no evidence that ciliary activity is reduced by the constituents of middle ear fluid in chronic otitis media with effusion.

TREX is a conserved complex coupling transcription with messenger RNA export.

The essential yeast proteins Yra1 and Sub2 are messenger RNA export factors that have conserved counterparts in metazoans, designated Aly and UAP56, respectively. These factors couple the machineries that function in splicing and export of mRNA. Here we show that both Yra1 and Sub2 are stoichiometrically associated with the heterotetrameric THO complex, which functions in transcription in yeast. We also show that Sub2 and Yra1 interact genetically with all four components of the THO complex (Tho2, Hpr1, Mft1 and Thp2). Moreover, these components operate in the export of bulk poly(A)(+) RNA as well as of mRNA derived from intronless genes. Both Aly and UAP56 associate with human counterparts of the THO complex. Together, these data define a conserved complex, designated the TREX ('transcription/export') complex. The TREX complex is specifically recruited to activated genes during transcription and travels the entire length of the gene with RNA polymerase II. Our data indicate that the TREX complex has a conserved role in coupling transcription to mRNA export.