Systematic Identification of H274Y Compensatory Mutations in Influenza A Virus Neuraminidase by High-Throughput Screening

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.     

Inference of the Genetic Architecture Underlying BMI and Height with the Use of 20,240 Sibling Pairs

Evidence that complex traits are highly polygenic has been presented by population-based genome-wide association studies (GWASs) through the identification of many significant variants, as well as by family-based de novo sequencing studies indicating that several traits have a large mutational target size. Here, using a third study design, we show results consistent with extreme polygenicity for body mass index (BMI) and height. On a sample of 20,240 siblings (from 9,570 nuclear families), we used a within-family method to obtain narrow-sense heritability estimates of 0.42 (SE = 0.17, p = 0.01) and 0.69 (SE = 0.14, p = 6 × 10⁻⁷) for BMI and height, respectively, after adjusting for covariates. The genomic inflation factors from locus-specific linkage analysis were 1.69 (SE = 0.21, p = 0.04) for BMI and 2.18 (SE = 0.21, p = 2 × 10⁻¹⁰) for height. This inflation is free of confounding and congruent with polygenicity, consistent with observations of ever-increasing genomic-inflation factors from GWASs with large sample sizes, implying that those signals are due to true genetic signals across the genome rather than population stratification. We also demonstrate that the distribution of the observed test statistics is consistent with both rare and common variants underlying a polygenic architecture and that previous reports of linkage signals in complex traits are probably a consequence of polygenic architecture rather than the segregation of variants with large effects. The convergent empirical evidence from GWASs, de novo studies, and within-family segregation implies that family-based sequencing studies for complex traits require very large sample sizes because the effects of causal variants are small on average.     

Emergence of an unrelated highly aberrant clone in an AML patient at relapse four months after peripheral blood stem cell transplantation

We report a case of AML-M1 with 5q aberration at diagnosis. The patient was treated with high-dose chemotherapy (HDCT). After remission induction, he received allogenic peripheral blood stem cell transplantation (PBSCT) from an HLA-match donor brother. The successive follow-up conventional cytogenetics investigations in remission after HDCT and PBSCT revealed cytogenetic remission. The most interesting observation in this case is that relapsed marrow revealed the emergence of an entirely new, highly aberrant, unrelated clone with unusual translocations t(6;17)(p23;p11.2),+8,der(8)dup inv(8)(q23qter), t(10;19)(q26;q13.3) 4½ months after PBSCT. Our findings suggest the possibility of a mutagenic effect of HDCT and myeloablative intense chemotherapy before PBSCT that could have induced a genetic lesion in the recipient''s genetically unstable stem cells in an environment of immunosuppression. The highly complex nature of the clone and the rapid clonal evolution indicates the possibility of selective pressure with proliferative advantage.     

Mucormycosis of the nasal ala in a leukemic (M7 AML) child. Is surgery of the nasal defect indicated?

Anterior tamponade with Surgicel (oxidized cellulose) was performed on a 5-year-old girl with megakaryoblastic leukemia (M7 AML) and epistaxis. Several days later necrosis of the nasal ala occurred. Debridement was performed and mucormycosis caused by Rhizopus was found in the material. Having cured mucormycosis, a defect of the complete nasal ala remained. The question of a surgical resolution of the disfiguring nasal defect arises.     

Mechanisms of Drosophila Dscam mutually exclusive splicing regulation

Alternative splicing of pre-mRNA is a major mechanism to increase protein diversity in higher eukaryotes. Dscam, the Drosophila homologue of human DSCAM (Down's syndrome cell adhesion molecule), generates up to 38016 isoforms through mutually exclusive splicing in four variable exon clusters. This enormous molecular diversity is functionally important for wiring of the nervous system and phagocytosis of invading pathogens. Current models explaining this complex splicing regulation include a default repressed state of the variable exon clusters to prevent the splicing together of adjacent exons, the presence of RNA secondary structures important for the release of one specific variable exon from the repressed state and combinatorial interaction of RNA-binding proteins for choosing a specific exon.     

Proteasomal inhibition sensitizes cervical cancer cells to mitomycin C-induced bystander effect: the role of tumor microenvironment

Inaccessibility of drugs to poorly vascularized strata of tumor is one of the limiting factors in cancer therapy. With the advent of bystander effect (BE), it is possible to perpetuate the cellular damage from drug-exposed cells to the unexposed ones. However, the role of infiltrating tumor-associated macrophages (TAMs), an integral part of the tumor microenvironment, in further intensifying BE remains obscure. In the present study, we evaluated the effect of mitomycin C (MMC), a chemotherapeutic drug, to induce BE in cervical carcinoma. By using cervical cancer cells and differentiated macrophages, we demonstrate that MMC induces the expression of FasL via upregulation of PPARγ in both cell types (effector cells) in vitro, but it failed to induce bystander killing in cervical cancer cells. This effect was primarily owing to the proteasomal degradation of death receptors in the cervical cancer cells. Pre-treatment of cervical cancer cells with MG132, a proteasomal inhibitor, facilitates MMC-mediated bystander killing in co-culture and condition medium transfer experiments. In NOD/SCID mice bearing xenografted HeLa tumors administered with the combination of MMC and MG132, tumor progression was significantly reduced in comparison with those treated with either agent alone. FasL expression was increased in TAMs, and the enhanced level of Fas was observed in these tumor sections, thereby causing increased apoptosis. These findings suggest that restoration of death receptor-mediated apoptotic pathway in tumor cells with concomitant activation of TAMs could effectively restrict tumor growth.Owing to the heterogeneous nature and scanty vascularization, the access of anticancer regimen to all strata of the tumor is one of the major challenges in cancer therapy. Current response rate to chemotherapy is far from desirable and warrants formulating the strategies to enhance specificity and efficacy of the anticancer regimens. Of late, the phenomenon of bystander effect (BE), which refers to transmission of death signals from the drug-exposed cells to the unexposed cells, is being explored to improve the therapeutic response. Although BE has been well documented in radiotherapy^{1, 2} and experimental approaches of gene therapy,^{3, 4} very limited information is available with respect to conventional chemotherapeutic drugs. We have previously demonstrated the chemotherapy-induced bystander killing in breast cancer cells⁵ and hepatocellular carcinoma cells.⁶ Recently, other groups also have demonstrated the occurrence of chemotherapy-induced BE in breast cancer⁷ and lung cancer,^{8, 9} which is in agreement with our studies. BE has been shown to be dependent on cell type and class of drugs,⁶ and the role of tumor microenvironment in response to chemotherapeutic drug-induced BE is poorly understood.Cervical cancer is one of the most common solid tumors. Mitomycin C (MMC), a DNA alkylating agent, has been widely used in this malignancy as a constituent of combination therapy.¹⁰ From the pharmacological point of view, MMC is effective at relatively low dose with minimal organ-associated toxicity¹¹ and it has been shown to activate innate immunity.¹² However, therapeutic efficacy of MMC principally depends on other drug types in combination therapy.¹³ Therefore, a well-designed strategy that could enhance the efficacy of MMC is desirable. MMC has been demonstrated to induce BE in hepatocellular carcinoma, but not in cervical cancer cells.⁶ Although the precise mechanisms of bystander killing remain elusive, we have previously reported the involvement of death ligands,^{5, 6} which was later supported by other studies.^{7, 8, 9} The ability of cancer cells to escape programmed cell death has a critical role in the survival of cancer cells and tumor progression. Despite the presence of cellular apoptotic factors, cancer cells reprogram their molecular events and signaling to evade apoptosis.¹⁴ Moreover, it has been reported that exposure to proteasomal inhibitor inhibits the growth of various cancer cells and sensitize them to death ligand-mediated death by stabilizing death receptors.^{15, 16, 17} Considering these notions, we speculated that non-functionality of death receptors could be one of the possible factors associated with defective BE in cervical cancer. We, therefore, hypothesized that treating cervical cancer cells with combination of MMC and proteasomal inhibitor could elicit BE, and thereby may significantly improve the therapeutic outcome.Till date, studies explicate cancer cells exposed to chemotherapy as the effector cells in inducing bystander-mediated killing. However, owing to the heterogeneous nature of cellular population in tumor, other cellular components are also likely to have a key role in inducing BE. Tumor microenvironment consists of a heterogeneous mass of malignant as well as nonmalignant cells. The nonmalignant cells include endothelial, fibroblast and immune cells that establish multitude of interactions among themselves and also with malignant cells.⁹ Macrophages are the most abundant immune cells present in tumors, also termed as tumor-associated macrophages (TAMs).¹⁸ TAMs are differentiated monocytes that infiltrate the tumor microenvironment, and are exposed to chemotherapeutic regimen. Studies have demonstrated that TAMs could account for approximately more than 60% of tumor mass in some cancers.^{19, 20, 21} TAMs exposed to radiations² and chemotherapy²² have been shown to have a significant role in inducing BE. Studies support the notion that targeting TAMs could improve the therapeutic index of various drugs.^{10, 23} Increased sensitivity to cyclophosphamide¹⁴ and cisplatin²⁴ has been shown in co-culture system involving cancer cells and macrophages. Under chemotherapy, increased recruitment of macrophages with enhanced expression of tumoricidal factors like perforin and granzyme,²² death ligands¹⁰ or ROS ²⁵ has been reported in tumors. Therefore, we speculated that BE could further be amplified by infiltrating macrophages resulting in enhanced therapeutic efficacy of anticancer regimens. In the present study, we evaluated combination effect of MMC and MG132 in enhancing bystander killing of cancer cells in vitro and in vivo, in part, through the involvement of cancer cells and TAMs. Herein, we demonstrate that stabilization of Fas on cervical cancer cells facilitates dramatic reduction in tumor progression as a consequence of increase in apoptosis. This study could be helpful in designing novel therapeutic strategies in treating cancer by involving proteasomal inhibitors in combination with chemotherapeutic drugs that specifically activate death receptor-mediated killing.     

Evaluating indirect genetic effects of siblings using singletons

Estimating effects of parental and sibling genotypes (indirect genetic effects) can provide insight into how the family environment influences phenotypic variation. There is growing molecular genetic evidence for effects of parental phenotypes on their offspring (e.g. parental educational attainment), but the extent to which siblings affect each other is currently unclear. Here we used data from samples of unrelated individuals, without (singletons) and with biological full-siblings (non-singletons), to investigate and estimate sibling effects. Indirect genetic effects of siblings increase (or decrease) the covariance between genetic variation and a phenotype. It follows that differences in genetic association estimates between singletons and non-singletons could indicate indirect genetic effects of siblings if there is no heterogeneity in other sources of genetic association between singletons and non-singletons. We used UK Biobank data to estimate polygenic score (PGS) associations for height, BMI and educational attainment in self-reported singletons (N = 50,143) and non-singletons (N = 328,549). The educational attainment PGS association estimate was 12% larger (95% C.I. 3%, 21%) in the non-singleton sample than in the singleton sample, but the height and BMI PGS associations were consistent. Birth order data suggested that the difference in educational attainment PGS associations was driven by individuals with older siblings rather than firstborns. The relationship between number of siblings and educational attainment PGS associations was non-linear; PGS associations were 24% smaller in individuals with 6 or more siblings compared to the rest of the sample (95% C.I. 11%, 38%). We estimate that a 1 SD increase in sibling educational attainment PGS corresponds to a 0.025 year increase in the index individual’s years in schooling (95% C.I. 0.013, 0.036). Our results suggest that older siblings may influence the educational attainment of younger siblings, adding to the growing evidence that effects of the environment on phenotypic variation partially reflect social effects of germline genetic variation in relatives.     

Voice rehabilitation of Broca's aphasia following total laryngectomy

Total laryngectomy, as a consequence of carcinoma of the larynx, results in loss of speech function. Cerebrovascular stroke is the leading cause of reduced speech production ability, and thereby communication difficulties. The case is presented of a 60-year-old male patient who suffered stroke five years after a total laryngectomy. Speech rehabilitation was hampered due to the depressive state of the patient. Although contraindicated, the secondary voice prosthesis was implanted. Only at that moment the patient showed willingness and motivation for speech rehabilitation. The aim of this presentation is to demonstrate that not all neurological disorders are contraindicated for implantation of voice prostheses.     

Systematic Review of Erythropoietin (EPO) for Neuroprotection in Human Studies

Erythropoietin (EPO) is an exciting neurotherapeutic option. Despite its potential, concerns exist regarding the potential for thrombosis and adverse events with EPO administration in normonemic adults. Systematic review of literature using PRISMA guidelines to examine the application and risks of EPO as a treatment option for neuroprotection in normonemic adults. Independent, systematic searches were performed in July 2019. PubMed (1960–2019) and the Cochrane Controlled Trials Register (1960–2019) were screened. Search terms included erythropoietin, neuroprotection, and humans. The PubMed search resulted in the following search strategy: (“erythropoietin” [MeSH Terms] OR “erythropoietin” [All Fields] OR “epoetin alfa” [MeSH Terms] OR (“epoetin” [All Fields] AND “alfa” [All Fields]) OR “epoetin alfa” [All Fields]) AND (“neuroprotection” [MeSH Terms] OR “neuroprotection” [All Fields]) AND “humans” [MeSH Terms]. PubMed, Cochrane Controlled Trials Register, and articles based on prior searches yielded 388 citations. 50 studies were included, comprising of 4351 patients. There were 13 studies that noted adverse effects from EPO. Three attributed serious adverse effects to EPO and complications were statistically significant. Two of these studies related the adverse events to the co-administration of EPO with tPA. Minor adverse effects associated with the EPO group included nausea, pyrexia, headache, generalized weakness and superficial phlebitis. Most published studies focus on spinal cord injury, peri-surgical outcomes and central effects of EPO. We found no studies to date evaluating the role of EPO in post-operative pain. Future trials could evaluate this application in persistent post-surgical pain and in the peri-operative period.