Human RECQL5 overcomes thymidine-induced replication stress

Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and γH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle.     

Relaxin-3 and receptors in the human and rhesus brain and reproductive tissues

Evidence suggests that relaxin-3 may have biological functions in the reproductive and central nervous systems. To date, however, relaxin-3 biodistribution has only been investigated in the mouse, rat, pig and teleost fish. Characterizing relaxin-3 gene structure, expression patterns, and function in non-human primates and humans is critical to delineating its biological significance. Experiments were performed to clone the rhesus macaque orthologues of the relaxin-3 peptide hormone and its cognitive receptors (RXFP1 and RXFP4). An investigation of rhesus relaxin-3 bioactivity and RXFP1 binding properties was also performed. Next we sought to investigate relaxin-3 immunoreactivity in human and rhesus macaque tissues. Immunohistofluorescence staining for relaxin-3 in the brain, testis, and prostate indicated predominant immunostaining in the ventral and dorsal tegmental nuclei, interstitial space surrounding the seminiferous tubules, and prostatic stromal cells, respectively. Further, in studies designed towards exploring biological functions, we observed neuroprotective actions of rhesus relaxin-3 on human neuronal cell cultures. Taken together, this study broadens the significance of relaxin-3 as a peptide involved in both neuronal cell function and reproductive tissues in primates.     

Regeneration of Leydig cells in unilaterally cryptorchid rats: evidence for stimulation by local testicular factors

Summary Leydig cells in testes of adult rats were selectively destroyed by a single intraperitoneal injection of ethane dimethane sulphonate. Four days later rats were made unilaterally cryptorchid and 1, 2 and 4 weeks later the histology of the testes was examined by light microscopy and morphometry. After induction of unilateral cryptorchidism, the volume of abdominal compared to scrotal testes was reduced by 45–60% due to rapid impairment of spermatogenesis in abdominal testes. Leydig cells were not present in either scrotal or abdominal testes in the 1-week unilateral crytorchid group. A new generation of foetal-type Leydig cells was observed in scrotal testes of the 2-week unilateral crytorchid group although their total volume per testis estimated by morphometry, was small, being approximately 1 l. In contrast, the abdominal testis exhibited a remarkable proliferation of foetal-type Leydig cells (total volume per testis, 16 l) which predominantly surrounded the peritubular tissues of the seminiferous tubules. A similar morphology and pattern of Leydig cell development was observed in scrotal and abdominal testes of the 4-week unilateral cryptorchid group where total Leydig cell volume was 7 l vs 21 l, respectively. The results show that regeneration of a new population of Leydig cells occurs more rapidly in the abdominal testis than in the scrotal testis of the same animal. These observations suggest the possibility that augmentation of Leydig cell growth is mediated by local intratesticular stimulatory factors within the abdominal testis. Development of new Leydig cells from the peritubular tissue provides circumstantial evidence that the seminiferous tubules and in particular the Sertoli cells, are a likely source of agents that stimulate the growth of Leydig cells.     

Presence of arbuscular mycorrhizas in typically ectomycorrhizal host species from Cameroon and New Zealand

 Ectomycorrhizas (EcM) and arbuscular mycorrhizas (AM) were screened for in saplings of 14 EcM tree species from the N'Dupé and Korup National Park rainforests, SW Cameroon, belonging to Caesalpiniaceae and Uapacaceae. The pattern of EcM and AM colonisation of a dual mycorrhizal species from this rainforest (Uapaca staudtii, Uapacaceae) was compared with dual EcM/AM colonisation of Leptospermum scoparium (Myrtaceae) from New Zealand. Both species were collected in a range of habitats. EcM and AM colonisation differed among species in the Korup National Park rainforest: 12 species belonging to the Caesalpiniaceae (Amherstieae) were consistently EcM, and AM structures occurred occasionally in six of them; two other species belonging to Caesalpiniaceae (Afzelia bipindensis) and Uapacaceae (U. staudtii) were dual mycorrhizal with variable levels of colonisation by both EcM and AM fungi. EcM and AM dual colonisation varied with both habitat and identity of the partners. The presence of EcM fungi in most of the root samples of U. staudtii and a negative relationship between AM and EcM colonisation within the same root system suggested a greater EcM affinity of this species. In contrast, most root samples of L. scoparium were colonised by AM, but only a few by EcM. Genuine dual EcM/AM associations in root samples of U. staudtii where the two mycorrhizal types co-occurred could be attributed to an AM-EcM succession. However, differences between predicted and observed frequencies of genuine dual EcM/AM associations in several samples of both U. staudtii and L. scoparium indicated that other factors influenced dual EcM/AM associations. The results of this study showed the importance of the identity of the host species in determining the pattern of dual EcM and AM colonisation. Accepted: 18 September 1998     

A Major Histocompatibility Class I Locus Contributes to Multiple Sclerosis Susceptibility Independently from HLA-DRB1*15:01

Background

In Northern European descended populations, genetic susceptibility for multiple sclerosis (MS) is associated with alleles of the human leukocyte antigen (HLA) Class II gene DRB1. Whether other major histocompatibility complex (MHC) genes contribute to MS susceptibility is controversial.

Methodology/Principal Findings

A case control analysis was performed using 958 single nucleotide polymorphisms (SNPs) spanning the MHC assayed in two independent datasets. The discovery dataset consisted of 1,018 cases and 1,795 controls and the replication dataset was composed of 1,343 cases and 1,379 controls. The most significantly MS-associated SNP in the discovery dataset was rs3135391, a Class II SNP known to tag the HLA-DRB1*15:01 allele, the primary MS susceptibility allele in the MHC (O.R. = 3.04, p<1×10⁻⁷⁸). To control for the effects of the HLA-DRB1*15:01 haplotype, case control analysis was performed adjusting for this HLA-DRB1*15:01 tagging SNP. After correction for multiple comparisons (false discovery rate = .05) 52 SNPs in the Class I, II and III regions were significantly associated with MS susceptibility in both datasets using the Cochran Armitage trend test. The discovery and replication datasets were merged and subjects carrying the HLA-DRB1*15:01 tagging SNP were excluded. Association tests showed that 48 of the 52 replicated SNPs retained significant associations with MS susceptibility independently of the HLA-DRB1*15:01 as defined by the tagging SNP. 20 Class I SNPs were associated with MS susceptibility with p-values ≤1×10⁻⁸. The most significantly associated SNP was rs4959039, a SNP in the downstream un-translated region of the non-classical HLA-G gene (Odds ratio 1.59, 95% CI 1.40, 1.81, p = 8.45×10⁻¹³) and is in linkage disequilibrium with several nearby SNPs. Logistic regression modeling showed that this SNP''s contribution to MS susceptibility was independent of the Class II and Class III SNPs identified in this screen.

Conclusions

A MHC Class I locus contributes to MS susceptibility independently of the HLA-DRB1*15:01 haplotype.     

Virus shedding kinetics and unconventional virulence tradeoffs

Tradeoff theory, which postulates that virulence provides both transmission costs and benefits for pathogens, has become widely adopted by the scientific community. Although theoretical literature exploring virulence-tradeoffs is vast, empirical studies validating various assumptions still remain sparse. In particular, truncation of transmission duration as a cost of virulence has been difficult to quantify with robust controlled in vivo studies. We sought to fill this knowledge gap by investigating how transmission rate and duration were associated with virulence for infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Using host mortality to quantify virulence and viral shedding to quantify transmission, we found that IHNV did not conform to classical tradeoff theory. More virulent genotypes of the virus were found to have longer transmission durations due to lower recovery rates of infected hosts, but the relationship was not saturating as assumed by tradeoff theory. Furthermore, the impact of host mortality on limiting transmission duration was minimal and greatly outweighed by recovery. Transmission rate differences between high and low virulence genotypes were also small and inconsistent. Ultimately, more virulent genotypes were found to have the overall fitness advantage, and there was no apparent constraint on the evolution of increased virulence for IHNV. However, using a mathematical model parameterized with experimental data, it was found that host culling resurrected the virulence tradeoff and provided low virulence genotypes with the advantage. Human-induced or natural culling, as well as host population fragmentation, may be some of the mechanisms by which virulence diversity is maintained in nature. This work highlights the importance of considering non-classical virulence tradeoffs.     

Dabrafenib; Preclinical Characterization,Increased Efficacy when Combined with Trametinib,while BRAF/MEK Tool Combination Reduced Skin Lesions

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAF^V600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G₁ cell cycle arrest, followed by cell death. In a BRAF^V600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor.     

Association between Apolipoprotein E genotype and cerebral palsy is not confirmed in a Caucasian population

Apolipoprotein E (APOE) plays a significant role in lipid metabolism and has been implicated in the growth and repair of injured neurons. Two small studies have suggested an association between APOE genotype and cerebral palsy. We investigated if APOE genotype is associated with an increased risk for cerebral palsy, influences the type of cerebral palsy or interacts with prenatal viral infection to influence risk of cerebral palsy. The population-based case-control study comprised newborn screening cards of 443 Caucasian patients with cerebral palsy and 883 Caucasian matched controls. APOE genotyping was performed on DNA extracted from dried blood spots. Allelic and genotypic frequencies did not differ between cases and controls and combined frequencies were 0.10 (ε2), 0.76 (ε3), 0.14 (ε4), 0.03 (ε2/ε2), 0.10 (ε2/ε3), 0.03 (ε2/ε4), 0.02 (ε4/ε4), 0.21 (ε3/ε4), 0.61 (ε3/ε3). APOE genotype was correlated with cerebral palsy, type of cerebral palsy, gestation at birth and the presence of viral nucleic acids detected in previous work. Analysis by gestational age (all gestational ages, ≥37, 32–36 and <32 weeks) and type of cerebral palsy (all types, diplegia, hemiplegia and quadriplegia) showed no association between APOE genotype and cerebral palsy in this Caucasian population. An association between prenatal viral infection, APOE genotype and cerebral palsy was not demonstrated. These results did not confirm an association between APOE genotype, cerebral palsy, type of cerebral palsy and prenatal infection in a Caucasian population. Given the low frequency of APOE ε2 and some of the heterozygote and homozygote combinations in this study, a larger study is assessing this further.