Human flap endonuclease structures, DNA double-base flipping, and a unified understanding of the FEN1 superfamily

Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5' flaps. FEN1 5' nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1), and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends, or Holliday junctions, respectively. Here, structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3' and 5' flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5' ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA; direct the target region into the helical gateway, permitting only nonbase-paired oligonucleotides active site access; and support a unified understanding of superfamily substrate specificity.     

Evidence that Cd101 is an autoimmune diabetes gene in nonobese diabetic mice

We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.     

Measuring the unknown: An estimator and simulation study for assessing case reporting during epidemics

The fraction of cases reported, known as ‘reporting’, is a key performance indicator in an outbreak response, and an essential factor to consider when modelling epidemics and assessing their impact on populations. Unfortunately, its estimation is inherently difficult, as it relates to the part of an epidemic which is, by definition, not observed. We introduce a simple statistical method for estimating reporting, initially developed for the response to Ebola in Eastern Democratic Republic of the Congo (DRC), 2018–2020. This approach uses transmission chain data typically gathered through case investigation and contact tracing, and uses the proportion of investigated cases with a known, reported infector as a proxy for reporting. Using simulated epidemics, we study how this method performs for different outbreak sizes and reporting levels. Results suggest that our method has low bias, reasonable precision, and despite sub-optimal coverage, usually provides estimates within close range (5–10%) of the true value. Being fast and simple, this method could be useful for estimating reporting in real-time in settings where person-to-person transmission is the main driver of the epidemic, and where case investigation is routinely performed as part of surveillance and contact tracing activities.     

Use of single nucleotide polymorphisms identifies backcrossing and species misidentifications among three San Francisco estuary osmerids

Two threatened osmerid species native to the San Francisco Estuary (SFE)—Delta Smelt (Hypomesus transpacificus) and Longfin Smelt (Spirinchus thaleichthys)—are subject to broad human influence, including significant habitat alteration and the presence of the introduced osmerid, Wakasagi (Hypomesus nipponensis). The identification of these closely related species and their hybrids is difficult in field collected specimens which are subject to damage through handling and may be difficult to identify morphologically, especially when young. In addition, it is known that these three species hybridize, but the extent and effect of hybridization is difficult to quantify and monitor. We developed assays for 24 species-specific single nucleotide polymorphisms (SNPs) that identify whether a sample is a pure species (Delta Smelt, Longfin Smelt, or Wakasagi), a first generation (F₁) hybrid, or a backcross. We used this SNP panel to genetically identify wild osmerids collected in Yolo Bypass from 2010 to 2016 and detected nine Delta Smelt × Wakasagi F₁ hybrids and two Wakasagi × (Delta Smelt × Wakasagi) backcross hybrids; all assayed hybrids had Wakasagi as the maternal parent. The backcrossing into Wakasagi suggests that hybridization may only occur in one direction and thus preclude introgression to Delta Smelt. We also found substantial morphological field misidentifications (32.7%) in the Yolo Bypass samples resulting in more Wakasagi and fewer Delta Smelt than previously recorded when based on morphology. The SNP panel described in this study constitutes a valuable resource for monitoring hybridization in the SFE and assigning species identifications with accuracy and efficiency.     

Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Background

The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50?kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

Methods

Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1?mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200?ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).

Results

The expected cDNA fragment of approximately 1240?bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10?mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.

Conclusions

The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

     

Temporal variation in constitutive and inducible heat shock proteins in the Zebra Finch Taeniopygia guttata

Cellular stressors initiate the heat shock response mediated by heat shock proteins (HSPs). There are two main types of HSPs, constitutive (always expressed) and inducible (upon stress), but as many in vivo studies fail to distinguish between them and because temporal expression patterns often differ among various types of HSPs, it is unclear when to measure HSPs. In this study, 26 (13 per treatment) adult female Zebra Finches Taeniopygia guttata were heat‐stressed (39 °C) or placed in a control brooder (room temperature) for 3 h and were bled 1 week prior to and at 1, 2, 4, 6 and 20 h post‐treatment. Treatment had no effect on levels of either constitutive HSP70 or inducible HSP90, but both HSPs decreased with time relative to baseline, suggesting a possible effect of handling stress and/or circadian variation.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

Superomedial pedicle technique of reduction mammaplasty

A series of 148 patients who underwent reduction mammaplasty utilizing the superomedial pedicle technique is presented. Resections as large as 4100 gm per breast with nipple-areola transpositions up to 30 cm were done with reliable nipple-areola survival, including preservation of sensation. The superior pedicle technique of breast reduction is recognized by many as technically easier and capable of producing a longer-lasting aesthetic effect. Classically, however, it has been limited to smaller resections. By incorporating the medial quadrant in the superior pedicle, more aggressive reductions can be safely undertaken with the same excellent results. Details of the procedure, the anatomic basis for its success, and complications are discussed.     

Spontaneous excitatory postsynaptic currents in crayfish neuromuscular junctions in the absence and presence of serotonin and 3,4-diaminopyridine

Summary Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded under voltage clamp in short fibres (l0.6mm) from opener muscles and the contractor epimeralis muscle of small crayfish. From the amplitude distributions of sEPSCs which could be approximated by a Gaussian function, a mean amplitudeã= –1.16 nA±0.28 (SE) was found for sEPSCs in 16 fibres of the claw opener voltage clamped toE=–60 mV (19–22 °C). In the opener of the first walking leg and in the contractor epimeralis muscleã=s-1.1 nA±0.21 (SE;n= 6, –100 mVE–60 mV, 5–10 °C) andã= –2.0 nA±0.2 (SE;n=4, E=–60 mV, 19–22 °C) were obtained. On average about 300–500 synaptic channels were estimated to open during a sEPSC. Giant sEPSCs (gsEPSCs) were also observed. The amplitudes of gsEPSCs were up to 14 times larger than the amplitude of an average normal sEPSC. Moreover, the lifetime of gsEPSCs was up to about 3 times longer than that of sEPSCs. Like sEPSCs, gsEPSCs could not be abolished by 0.1 mol/l tetrodotoxin. The rate at which sEPSCs and gsEPSCs occurred could be markedly enhanced by serotonin (1 mol/l) and 3,4-diaminopyridine (1 mol/l)Abbreviations sEPSCs spontaneous excitatory postsynaptic currents - gsEPSCs giant spontaneous excitatory postsynaptic currents - sIPSCs spontaneous inhibitory postsynaptic currents - gsIPSCs giant spontaneous inhibitory postsynaptic currents - 5-HT 5-hydroxytryptamine - 3,4-DAP 3,4-diaminopyridine - time constant of exponential decay of sEPSCs or gsEPSCs - t _B50 lifetime of sEPSCs or gsEPSCs given by the width of these currents at 50% of their amplitude; ã amplitude of sEPSCs or gsEPSCs - i current amplitude evoked by opening of single glutamate-activated channels - z number of channels open at the peak of an average sEPSC This investigation was supported by the Deutsche Forschungsgemeinschaft. Project Fi 305/1-3