Alloimmune response results in expansion of autoreactive donor CD4+ T cells in transplants that can mediate chronic graft-versus-host disease

Chronic graft-versus-host disease (cGVHD) is considered an autoimmune-like disease mediated by donor CD4(+) T cells, but the origin of the autoreactive T cells is still controversial. In this article, we report that the transplantation of DBA/2 donor spleen cells into thymectomized MHC-matched allogeneic BALB/c recipients induced autoimmune-like cGVHD, although not in control syngeneic DBA/2 recipients. The donor-type CD4(+) T cells from the former but not the latter recipients induced autoimmune-like manifestations in secondary allogeneic BALB/c as well as syngeneic DBA/2 recipients. Transfer of donor-type CD4(+) T cells from secondary DBA/2 recipients with disease into syngeneic donor-type or allogeneic host-type tertiary recipients propagated autoimmune-like manifestations in both. Furthermore, TCR spectratyping revealed that the clonal expansion of the autoreactive CD4(+) T cells in cGVHD recipients was initiated by an alloimmune response. Finally, hybridoma CD4(+) T clones derived from DBA/2 recipients with disease proliferated similarly in response to stimulation by syngeneic donor-type or allogeneic host-type dendritic cells. These results demonstrate that the autoimmune-like manifestations in cGVHD can be mediated by a population of donor CD4(+) T cells in transplants that simultaneously recognize Ags presented by both donor and host APCs.     

Carbon oxidation and incorporation patterns in batch cultures ofMethylomonas L3

Summary A recently developed methodology of directly measuring the oxidation and incorporation patterns of carbon substrate in continuous cultures of RuMP-type methylotrophs is extended to batch cultures of the obligate methylotrophMethylomonas L3. The ratio of cyclic to total substrate oxidation varies with the initial methanol concentration from 0 to 68%. Formaldehyde, as a methanol cosubstrate, enhances the net substrate oxidation. The substrate oxidation and incorporation pattern is also affected by the state of the culture inoculum.     

Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method

Good protein thermostability is very important for the protein application. In this report, we propose a strategy which contained a prediction method to select residues related to protein thermal stability, but not related to protein function, and an experiment method to screen the mutants with enhanced thermostability. The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH were selected and used to construct seven saturation mutagenesis libraries. The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme. A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations (S274Q/T183E/K197L/S192M) was selected. The T _m and T ₅₀ of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering.     

CONSPECIFIC SPERM PRECEDENCE IS AN EFFECTIVE BARRIER TO HYBRIDIZATION BETWEEN CLOSELY RELATED SPECIES

Conspecific sperm precedence is widespread in animals, appears to evolve rapidly, and is thought to have the potential to prevent hybridization between closely related species. However, to date no study has tested the isolating potential of such a barrier in mixed populations of two taxa under conditions in which other potential barriers to gene flow are controlled for or are prevented from operating. We tested the isolating potential of conspecific sperm precedence in the ground crickets Allonemobius fasciatus and A. socius in population cage experiments in which the frequency of the two species was varied. Despite the observation of abundant interspecific matings, the proportions of hybrid progeny were low and differed statistically from the proportions expected in the absence of conspecific sperm precedence. The results demonstrate that conspecific sperm precedence can severely limit gene flow between closely related species, even when one species is less abundant than the other.     

The accumulation of chromosome aberrations and Dlb-1 mutations in mice with highly fractionated exposure to gamma radiation

The dichotomy between the doses at which experimental measurements of genetic effects can be made and the doses to which people are exposed is often different by two or more orders of magnitude. This presents a significant problem when determining the effects of low doses of radiation or chemicals. The solution has usually involved extrapolating the data by curve-fitting or by applying theoretical considerations. Both approaches are unsatisfactory due to uncertainties of the assumptions used in each process. The alternative solution has been to increase the sample size enormously at the lower doses. This is impractical beyond a certain point due to the variation in the spontaneous frequency and the need to quadruple the sample size for a doubling of precision. The development of new methods for measuring stable genetic effects, however, permits a simple and effective approach to this problem: if the genetic events being detected have no effect on survival, i.e., are selectively neutral, then the effects of multiple independent treatments will be additive. If the independent treatments are identical, then the effect of each is easily calculated by dividing the total effect by the number of treatments. Here we report a limited test of this approach using mice. Chromosome aberrations induced in lymphocytes and Dlb-1 mutations induced in the small intestine were measured after daily doses of 0.64, 1.85 or 5.5 cGy ¹³⁷Cs gamma rays administered for 21, 42 or 63 days. The dose response curve for chromosome translocations obtained in this way, combined with the data from single larger acute doses, shows no evidence for a threshold over a 500-fold dose range. Dlb-1 mutations were increased at each dose and time but the results do not permit reliable extrapolations. The results suggest that translocations might be useful for quantifying the effect of doses below 0.05 cGy and that the effect of dose rate and dose fractionation at much lower doses than reported here could be investigated.     

Overexpression of hCLP46 enhances Notch activation and regulates cell proliferation in a cell type‐dependent manner

Objectives

Human CAP10‐like protein 46 kDa (hCLP46), also known as Poglut1, has been shown to be an essential regulator of Notch signalling. hCLP46 is overexpressed in primary acute myelogenous leukaemia, T‐acute lymphoblastic leukaemia samples and other leukaemia cell lines. However, effects of hCLP46 overexpression, up to now, have remained unknown.

Materials and methods

In this study, we established stable 293TRex cell lines inducibly overexpressing hCLP46, and knocked down hCLP6 with a specific small interfering RNA to explore function of the protein in Notch signalling and cell proliferation.

Results

hCLP46 overexpression enhanced Notch1 activation in 293Trex cells in a ligand‐dependent manner, with increased Notch signalling enhancing Hes1 expression. We further verified that overexpression of hCLP46 inhibited proliferation of 293TRexs and was correlated with increases in cyclin dependent kinase inhibitors p21 and p27, whereas reduced hCLP46 expression moderately increased cell proliferation. In addition, p21 and p27 protein levels were higher when Notch signalling was activated by EDTA treatment.

Conclusions

Taken together, hCLP46 enhanced Notch activation and inhibited 293TRex cell proliferation through CDKI signalling.

     

A sensitive,specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivax and Plasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivax and Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed with Plasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochrome b-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.     

Quantifying multiple pressure interactions affecting populations of a recreationally and commercially important freshwater fish

The expanding human global footprint and growing demand for freshwater have placed tremendous stress on inland aquatic ecosystems. Aichi Target 10 of the Convention on Biological Diversity aims to minimize anthropogenic pressures affecting vulnerable ecosystems, and pressure interactions are increasingly being incorporated into environmental management and climate change adaptation strategies. In this study, we explore how climate change, overfishing, forest disturbance, and invasive species pressures interact to affect inland lake walleye (Sander vitreus) populations. Walleye support subsistence, recreational, and commercial fisheries and are one of most sought‐after freshwater fish species in North America. Using data from 444 lakes situated across an area of 475 000 km² in Ontario, Canada, we apply a novel statistical tool, R‐INLA, to determine how walleye biomass deficit (carrying capacity—observed biomass) is impacted by multiple pressures. Individually, angling activity and the presence of invasive zebra mussels (Dreissena polymorpha) were positively related to biomass deficits. In combination, zebra mussel presence interacted negatively and antagonistically with angling activity and percentage decrease in watershed mature forest cover. Velocity of climate change in growing degree days above 5°C and decrease in mature forest cover interacted to negatively affect walleye populations. Our study demonstrates how multiple pressure evaluations can be conducted for hundreds of populations to identify influential pressures and vulnerable ecosystems. Understanding pressure interactions is necessary to guide management and climate change adaptation strategies, and achieve global biodiversity targets.     

Prolonged Cerebral Circulation Time Is the Best Parameter for Predicting Vasospasm during Initial CT Perfusion in Subarachnoid Hemorrhagic Patients

Purpose

We sought to imitate angiographic cerebral circulation time (CCT) and create a similar index from baseline CT perfusion (CTP) to better predict vasospasm in patients with subarachnoid hemorrhage (SAH).

Methods

Forty-one SAH patients with available DSA and CTP were retrospectively included. The vasospasm group was comprised of patients with deterioration in conscious functioning and newly developed luminal narrowing; remaining cases were classified as the control group. The angiography CCT (XA-CCT) was defined as the difference in TTP (time to peak) between the selected arterial ROIs and the superior sagittal sinus (SSS). Four arterial ROIs were selected to generate four corresponding XA-CCTs: the right and left anterior cerebral arteries (XA-CCT_RA2 and XA-CCT_LA2) and right- and left-middle cerebral arteries (XA-CCT_RM2 and XA-CCT_LM2). The CCTs from CTP (CT-CCT) were defined as the differences in TTP from the corresponding arterial ROIs and the SSS. Correlations of the different CCTs were calculated and diagnostic accuracy in predicting vasospasm was evaluated.

Results

Intra-class correlations ranged from 0.96 to 0.98. The correlations of XA-CCT_RA2, XA-CCT_RM2, XA-CCT_LA2, and XA-CCT_LM2 with the corresponding CT-CCTs were 0.64, 0.65, 0.53, and 0.68, respectively. All CCTs were significantly prolonged in the vasospasm group (5.8–6.4 s) except for XA-CCT_LA2. CT-CCT_A2 of 5.62 was the optimal cut-off value for detecting vasospasm with a sensitivity of 84.2% and specificity 82.4%

Conclusion

CT-CCTs can be used to interpret cerebral flow without deconvolution algorithms, and outperform both MTT and TTP in predicting vasospasm risk. This finding may help facilitate management of patients with SAH.