The Epstein-Barr Virus Glycoprotein BDLF2 Is Essential for Efficient Viral Spread in Stratified Epithelium.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen that infects the majority of the adult population regardless of socioeconomic status or geographical location. EBV primarily infects B and epithelial cells and is associated with different cancers of these cell types, such as Burkitt lymphoma and nasopharyngeal carcinoma. While the life cycle of EBV in B cells is well understood, EBV infection within epithelium is not, largely due to the inability to model productive replication in epithelium in vitro. Organotypic cultures generated from primary human keratinocytes can model many aspects of EBV infection, including productive replication in the suprabasal layers. The EBV glycoprotein BDLF2 is a positional homologue of the murine gammaherpesvirus-68 protein gp48, which plays a role in intercellular spread of viral infection, though sequence homology is limited. To determine the role that BDLF2 plays in EBV infection, we generated a recombinant EBV in which the BDLF2 gene has been replaced with a puromycin resistance gene. The ΔBDLF2 recombinant virus infected both B cell and HEK293 cell lines and was able to immortalize primary B cells. However, the loss of BDLF2 resulted in substantially fewer infected cells in organotypic cultures compared to wild-type virus. While numerous clusters of infected cells representing a focus of infection are observed in wild-type-infected organotypic cultures, the majority of cells observed in the absence of BDLF2 were isolated cells, suggesting that the EBV glycoprotein BDLF2 plays a major role in intercellular viral spread in stratified epithelium. IMPORTANCE The ubiquitous herpesvirus Epstein-Barr virus (EBV) is associated with cancers of B lymphocytes and epithelial cells and is primarily transmitted in saliva. While several models exist for analyzing the life cycle of EBV in B lymphocytes, models of EBV infection in the epithelium have more recently been established. Using an organotypic culture model of epithelium that we previously determined accurately reflects EBV infection in situ, we have ascertained that the loss of the viral envelope protein BDLF2 had little effect on the EBV life cycle in B cells but severely restricted the number of infected cells in organotypic cultures. Loss of BDLF2 has a substantial impact on the size of infected areas, suggesting that BDLF2 plays a specific role in the spread of infection in stratified epithelium.

Assessing nonsexual transmission of the human papillomavirus (HPV): Do our current cleaning methods work?

Human papillomavirus (HPV) is thought to be sexually transmitted; however, there have been a few studies investigating a possible iatrogenic source of infection. Therefore, it is important to assess the cleaning methods of reusable medical devices. This study assessed whether cleaning methods of flexible endoscopes in an otolaryngology clinic are effective against HPV. There were 24 patients with a history of head and neck cancer in the study; however, two outliers were excluded. Nine patients were confirmed to have HPV-associated cancer. PCR was used to measure and quantify the viral genomes of samples collected before and after cleaning. After cleaning, few HPV+ samples had endoscopes with less DNA than before cleaning. Additionally, for several patients with non-HPV-associated head and neck cancer, PCR showed more DNA after cleaning than before cleaning, suggesting residual HPV DNA within the cleaning solution. There was no significant difference (p > 0.05) between pre- and post-cleaning in both cohorts. Current cleaning methods of reusable endoscopes may not be effective in completely removing viral DNA.

Hypochlorous acid as a disinfectant for high-risk HPV: Insight into the mechanism of action.

Medical instruments that are not autoclavable but may become contaminated with high-risk human papillomaviruses (HPVs) during use must be thoroughly disinfected to avoid the possibility of iatrogenic transmission of infection. There is an expectation that prolonged soaking of instruments in the United States Food and Drug Administration-cleared chemical disinfectant solutions will result in high-level decontamination, but HPV16 and HPV18 are known to be resistant to commonly used formulations. However, they are susceptible to a variety of oxidative agents, including those based on chlorine. Here, we tested the efficacy of homogeneous hypochlorous acid (HOCl) solutions against mature infectious virions of HPV16 and HPV18 dried onto butadiene styrene coupons and ultrasonic probes. Both viruses were inactivated to >4 log reduction value (LRV) after 15 s on coupons and 5 min on ultrasonic probes. Morphologic changes became evident within those contact times by transmission electron microscopy when HPV16 virus-like particles were exposed to HOCl under identical conditions. Mass spectrometry analysis of trypsin-digested products of L1 capsid proteins exposed to HOCl showed that mostly conserved residues were modified by oxidation and that these changes rapidly lead to instability of the protein demonstrable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Modifications to these residues may contribute to rapid virus inactivation. The use of homogeneous HOCl solutions for HPV decontamination provides a highly effective means of assuring the safety of nonautoclavable medical instruments.

Lowering the transmission and spread of human coronavirus.

The emergence of the severe acute respiratory syndrome coronavirus 2 pandemic has created an unprecedented healthcare, social, and economic disaster. Wearing of masks and social distancing can significantly decrease transmission and spread, however, due to circumstances such as medical or dental intervention and personal choice these practices have not been universally adopted. Additional strategies are required to lessen transmission. Nasal rinses and mouthwashes, which directly impact the major sites of reception and transmission of human coronaviruses (HCoV), may provide an additional level of protection against the virus. Common over-the-counter nasal rinses and mouthwashes/gargles were tested for their ability to inactivate high concentrations of HCoV using contact times of 30 s, 1 min, and 2 min. Reductions in titers were measured by using the tissue culture infectious dose 50 (TCID50 ) assay. A 1% baby shampoo nasal rinse solution inactivated HCoV greater than 99.9% with a 2-min contact time. Several over-the-counter mouthwash/gargle products including Listerine and Listerine-like products were highly effective at inactivating infectious virus with greater than 99.9% even with a 30-s contact time. In the current manuscript we have demonstrated that several commonly available healthcare products have significant virucidal properties with respect to HCoV.

Tissue-Specific Gene Expression during Productive Human Papillomavirus 16 Infection of Cervical, Foreskin, and Tonsil Epithelium.

Epidemiological data confirm a much higher incidence of high-risk human papillomavirus 16 (HPV16)-mediated carcinogenesis of the cervical epithelium than for other target sites. In order to elucidate tissue-specific responses to virus infection, we compared gene expression changes induced by productive HPV16 infection of cervical, foreskin, and tonsil organotypic rafts. These rafts closely mimic persistent HPV16 infection, long before carcinogenesis sets in. The total number of gene expression changes varied considerably across the tissue types, with only 32 genes being regulated in common. Among them, we confirmed the Kelch-like family protein KLHL35 and the laminin-5 complex to be upregulated and downregulated, respectively, in all the three tissues. HPV16 infection induces upregulation of genes involved in cell cycle control, cell division, mitosis, DNA replication, and DNA damage repair in all the three tissues, indicative of a hyperproliferative environment. In the cervical and tonsil epithelium, we observe significant downregulation of genes involved in epidermis development, keratinocyte differentiation, and extracellular matrix organization. On the other hand, in HPV16-positive foreskin (HPV16 foreskin) tissue, several genes involved in interferon-mediated innate immunity, cytokine signaling, and cellular defenses were downregulated. Furthermore, pathway analysis and experimental validations identified important cellular pathways like STAT1 and transforming growth factor ß (TGF-ß) to be differentially regulated among the three tissue types. The differential modulation of important cellular pathways like TGF-ß1 and STAT1 can explain the sensitivity of tissues to HPV cancer progression.IMPORTANCE Although the high-risk human papillomavirus 16 infects anogenital and oropharyngeal sites, the cervical epithelium has a unique vulnerability to progression of cancer. Host responses during persistent infection and preneoplastic stages can shape the outcome of cancer progression in a tissue-dependent manner. Our study for the first time reports differential regulation of critical cellular functions and signaling pathways during productive HPV16 infection of cervical, foreskin, and tonsil tissues. While the virus induces hyperproliferation in infected cells, it downregulates epithelial differentiation, epidermal development, and innate immune responses, according to the tissue type. Modulation of these biological functions can determine virus fitness and pathogenesis and illuminate key cellular mechanisms that the virus employs to establish persistence and finally initiate disease progression.

Effect of Productive Human Papillomavirus 16 Infection on Global Gene Expression in Cervical Epithelium.

Human papillomavirus (HPV) infection is the world's most common sexually transmitted infection and is responsible for most cases of cervical cancer. Previous studies of global gene expression changes induced by HPV infection have focused on the cancerous stages of infection, and therefore, not much is known about global gene expression changes at early preneoplastic stages of infection. We show for the first time the global gene expression changes during early-stage HPV16 infection in cervical tissue using 3-dimensional organotypic raft cultures, which produce high levels of progeny virions. cDNA microarray analysis showed that a total of 594 genes were upregulated and 651 genes were downregulated at least 1.5-fold with HPV16 infection. Gene ontology analysis showed that biological processes including cell cycle progression and DNA metabolism were upregulated, while skin development, immune response, and cell death were downregulated with HPV16 infection in cervical keratinocytes. Individual genes were selected for validation at the transcriptional and translational levels, including UBC, which was central to the protein association network of immune response genes, and top downregulated genes RPTN, SERPINB4, KRT23, and KLK8 In particular, KLK8 and SERPINB4 were shown to be upregulated in cancer, which contrasts with the gene regulation during the productive replication stage. Organotypic raft cultures, which allow full progression of the HPV life cycle, allowed us to identify novel gene modulations and potential therapeutic targets of early-stage HPV infection in cervical tissue. Additionally, our results suggest that early-stage productive infection and cancerous stages of infection are distinct disease states expressing different host transcriptomes.IMPORTANCE Persistent HPV infection is responsible for most cases of cervical cancer. The transition from precancerous to cancerous stages of HPV infection is marked by a significant reduction in virus production. Most global gene expression studies of HPV infection have focused on the cancerous stages. Therefore, little is known about global gene expression changes at precancerous stages. For the first time, we measured global gene expression changes at the precancerous stages of HPV16 infection in human cervical tissue producing high levels of virus. We identified a group of genes that are typically overexpressed in cancerous stages to be significantly downregulated at the precancerous stage. Moreover, we identified significantly modulated genes that have not yet been studied in the context of HPV infection. Studying the role of these genes in HPV infection will help us understand what drives the transition from precancerous to cancerous stages and may lead to the development of new therapeutic targets.

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation.

The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.

Subtype-specific accumulation of intracellular zinc pools is associated with the malignant phenotype in breast cancer.

BACKGROUND: Zinc (Zn) hyper-accumulates in breast tumors and malignant cell lines compared to normal mammary epithelium. The mechanisms responsible for Zn accumulation and the consequence of Zn dysregulation are poorly understood. METHODS: Microarrays were performed to assess differences in the expression of Zn transporters and metallothioneins (MTs) in human breast tumors and breast cancer cell lines. Real-time PCR and immunoblotting were employed to profile Zn transporter expression in representative luminal (T47D), basal (MDA-MB-231), and non-malignant (MCF10A) cell lines. Zn distribution in human tumors was assessed by X-ray fluorescence imaging. Zn distribution and content in cell lines was measured using FluoZin-3 imaging, and quantification and atomic absorption spectroscopy. Functional consequences of ZnT2 over-expression in MDA-MB-231 cells including invasion, proliferation, and cell cycle were measured using Boyden chambers, MTT assays, and flow cytometry, respectively. RESULTS: Gene expression profiling of human breast tumors and breast cancer cell lines identified subtype-specific dysregulation in the Zn transporting network. X-ray fluorescence imaging of breast tumor tissues revealed Zn hyper-accumulation at the margins of Luminal breast tumors while Zn was more evenly distributed within Basal tumors. While both T47D and MDA-MB-231 cells hyper-accumulated Zn relative to MCF10A cells, T47D cells accumulated 2.5-fold more Zn compared to MDA-MB-231 cells. FluoZin-3 imaging indicated that Zn was sequestered into numerous large vesicles in T47D cells, but was retained in the cytoplasm and found in fewer and larger, amorphous sub-cellular compartments in MDA-MB-231 cells. The differences in Zn localization mirrored the relative abundance of the Zn transporter ZnT2; T47D cells over-expressed ZnT2, whereas MDA-MB-231 cells did not express ZnT2 protein due to proteasomal degradation. To determine the functional relevance of the lack of ZnT2 in MDA-MB-231cells, cells were transfected to express ZnT2. ZnT2 over-expression led to Zn vesicularization, shifts in cell cycle, enhanced apoptosis, and reduced proliferation and invasion. CONCLUSIONS: This comprehensive analysis of the Zn transporting network in malignant breast tumors and cell lines illustrates that distinct subtype-specific dysregulation of Zn management may underlie phenotypic characteristics of breast cancers such as grade, invasiveness, metastatic potential, and response to therapy.

Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction.

The zinc (Zn) transporter ZnT2 (SLC30A2) is expressed in specialized secretory cells including breast, pancreas and prostate, and imports Zn into mitochondria and vesicles. Mutations in SLC30A2 substantially reduce milk Zn concentration ([Zn]) and cause severe Zn deficiency in exclusively breastfed infants. Recent studies show that ZnT2-null mice have low milk [Zn], in addition to profound defects in mammary gland function during lactation. Here, we used breast milk [Zn] to identify novel non-synonymous ZnT2 variants in a population of lactating women. We also asked whether specific variants induce disturbances in intracellular Zn management or cause cellular dysfunction in mammary epithelial cells. Healthy, breastfeeding women were stratified into quartiles by milk [Zn] and exonic sequencing of SLC30A2 was performed. We found that 36% of women tested carried non-synonymous ZnT2 variants, all of whom had milk Zn levels that were distinctly above or below those in women without variants. We identified 12 novel heterozygous variants. Two variants (D(103)E and T(288)S) were identified with high frequency (9 and 16%, respectively) and expression of T(288)S was associated with a known hallmark of breast dysfunction (elevated milk sodium/potassium ratio). Select variants (A(28)D, K(66)N, Q(71)H, D(103)E, A(105)P, Q(137)H, T(288)S and T(312)K) were characterized in vitro. Compared with wild-type ZnT2, these variants were inappropriately localized, and most resulted in either 'loss-of-function' or 'gain-of-function', and altered sub-cellular Zn pools, Zn secretion, and cell cycle check-points. Our study indicates that SLC30A2 variants are common in this population, dysregulate Zn management and can lead to breast cell dysfunction. This suggests that genetic variation in ZnT2 could be an important modifier of infant growth/development and reproductive health/disease. Importantly, milk [Zn] level may serve as a bio-reporter of breast function during lactation.

Human papillomavirus (HPV) upregulates the cellular deubiquitinase UCHL1 to suppress the keratinocyte's innate immune response.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Prolactin receptor attenuation induces zinc pool redistribution through ZnT2 and decreases invasion in MDA-MB-453 breast cancer cells.

Prolactin receptor (PRL-R) activation regulates cell differentiation, proliferation, cell survival and motility of breast cells. Prolactin (PRL) and PRL-R over-expression are strongly implicated in breast cancer, particularly contributing to tumor growth and invasion in the more aggressive estrogen-receptor negative (ER-) disease. PRL-R antagonists have been suggested as potential therapeutic agents; however, mechanisms through which PRL-R antagonists exert their actions are not well-understood. Zinc (Zn) is a regulatory factor for over 10% of the proteome, regulating critical cell processes such as proliferation, cell signaling, transcription, apoptosis and autophagy. PRL-R signaling regulates Zn metabolism in breast cells. Herein we determined effects of PRL-R attenuation on cellular Zn metabolism and cell function in a model of ER-, PRL-R over-expressing breast cancer cells (MDA-MB-453). PRL-R attenuation post-transcriptionally increased ZnT2 abundance and redistributed intracellular Zn pools into lysosomes and mitochondria. ZnT2-mediated lysosomal Zn sequestration was associated with reduced matrix metalloproteinase 2 (MMP-2) activity and decreased invasion. ZnT2-mediated Zn accumulation in mitochondria was associated with increased mitochondrial oxidation. Our results suggest that PRL-R antagonism in PRL-R over-expressing breast cancer cells may reduce invasion through the redistribution of intracellular Zn pools critical for cellular function.

Techno-economic and environmental analysis of organic municipal solid waste for energy production.

Addressing the critical conundrum of escalating municipal solid waste (MSW) and shrinking landfill spaces in urban areas, this research pioneers a sustainable approach for Bangladesh by exploring the potential of biogas production from MSW. Distinctly, it fills the research gap by providing a detailed techno-economic and environmental analysis of decentralized fixed-dome anaerobic digestion facilities in the urban context of Chittagong, Bangladesh, a domain previously underexplored. Our findings demonstrate the feasibility of converting MSW into a renewable energy source, offering an innovative solution that simultaneously tackles waste management and energy generation challenges. Each proposed plant showcases the capability to generate 536 m³ of biogas daily, sufficient to power a 50 kW gas engine and supply 44 households, thereby contributing significantly to urban waste reduction and CO2 emissions mitigation by approximately 500 tons monthly. The economic analysis reveals an attractive investment payback period of two years, underscoring the model's viability and its potential as a replicable framework for similar urban settings grappling with waste management crises. This study not only bridges a critical knowledge gap but also introduces a novel, sustainable waste-to-energy model, marking a pivotal step towards achieving energy security and environmental sustainability in developing nations.

Diversity in Proprotein Convertase Reactivity among Human Papillomavirus Types.

The cleavage of viral surface proteins by furin is associated with some viruses' high virulence and infectivity. The human papillomavirus (HPV) requires the proteolytic processing of its capsid proteins for activation before entry. Variability in reactivity with furin and other proprotein convertases (PCs) among HPV types was investigated. HPV16, the most prevalent and carcinogenic HPV type, reacted with PCs with the broadest selectivity compared to other types in reactions of pseudoviral particles with the recombinant PCs, furin, PC4, PC5, PACE4, and PC7. Proteolytic preactivation was assessed using a well-established entry assay into PC-inhibited cells based on the green fluorescent protein as a reporter. The inhibition of the target cell PC activity with serpin-based PC-selective inhibitors also showed a diversity of PC selectivity among HPV types. HPV16 reacted with furin at the highest rate compared to the other types in time-dependent preactivation reactions and produced the highest entry values standardized to pseudoviral particle concentration. The predominant expression of furin in keratinocytes and the high reactivity of HPV16 with this enzyme highlight the importance of selectively targeting furin as a potential antiviral therapeutic approach.

Treatment of a human papillomavirus type 31b-positive cell line with benzo[a]pyrene increases viral titer through activation of the Erk1/2 signaling pathway.

Numerous epidemiological studies have implicated cigarette smoking as a cofactor in the progression to cervical cancer. Tobacco-associated hydrocarbons have been found in cervical mucus, suggesting a possible interaction with human papillomavirus (HPV)-infected cells. The polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) is a major component of cigarette smoke condensate that has received significant attention due to its ability to induce carcinogenesis. We have previously demonstrated by conventional methods for determining viral titer that high concentrations of BaP increase HPV31b titers within the context of organotypic raft cultures compared with the level for vehicle controls. However, a definitive mechanism for explaining this increase in viral titer was lacking. Here, we show that BaP treatment activates the Ras-Raf-Mek1/2-Erk1/2 signaling pathway. The importance of Erk1/2 pathway activation to the BaP-mediated increase in viral titer was determined by Erk pathway inhibition with multiple Erk1/2 pathway inhibitors. Finally, BaP treatment activated p90RSK and its downstream target CDK1. These data indicate that the Erk1/2 signaling pathway plays an important role in mediating the response to BaP treatment that ultimately leads to increased viral titers.

Comparative metabolism of benzo[a]pyrene by human keratinocytes infected with high-risk human papillomavirus types 16 and 18 as episomal or integrated genomes.

BACKGROUND: Infection with human papillomavirus (HPV) is a critical factor in the development of cervical cancer. Smoking is an additional risk factor. Tobacco smoke carcinogens, such as benzo[a]pyrene (B[a]P), and their cytochrome P450-related metabolites are present in significantly higher levels in the cervical mucus of women smokers than in nonsmokers. We determined the metabolism and P450 expression of B[a]P-treated human keratinocytes infected with HPV-16 or -18. MATERIALS AND METHODS: Monolayer cultures of uninfected primary human foreskin keratinocytes, human vaginal and cervical keratinocytes carrying episomal genomes of HPV-16 and -18, respectively, and invasive cervical carcinoma cell lines carrying either HPV-16 or -18 genomes integrated into the host DNA, were incubated with 0.1 µM [(3)H]B[a]P. The resulting oxidative metabolites were analyzed and quantified by radioflow high-performance liquid chromatography. Additionally, all cell lines were incubated with unlabeled 0.1 µM B[a]P for Western blot analysis of cytochrome P450 1A1 and 1B1. RESULTS: Significant enhancement in levels of both detoxification and activation metabolites was found in incubations with all types of HPV-infected cells compared with control incubations (P < 0.05). The highest capacity to metabolize B[a]P was observed with cells containing integrated HPV-18 genomes. Induction of cytochrome 1B1 was observed in HPV-16 and -18 integrated, and in HPV-16 episomal cell types. CONCLUSIONS: Both viral genotype and genomic status in the host cell affect B[a]P metabolism and cytochrome P450 1B1 expression. An increase of DNA-damaging metabolites might result from exposure of HPV-infected women to cigarette smoke carcinogens.

Cis-diastereoselectivity in pictet-spengler reactions of L-tryptophan and electronic circular dichroism studies.

The diastereoselective synthesis of optically active 1,3-disubstituted tetrahydro-ß-carbolines using polar protic Pictet-Spengler cyclization of (S)-tryptophan methyl ester with five aldehydes RCHO (R═CH(3), C(2)H(5), C(3)H(7), C(4)H(9), and C(6)H(5)) was studied. As an alternate route, the cyclization of (S)-tryptophan with the same aldehydes and subsequent methylation of the resulting tetrahydro-ß-carboline carboxylic acids were also performed for comparison. (13)C NMR and electronic circular dichroism (ECD) studies and time-dependent density functional theory ECD calculations data established the relative 1,3 cis/trans and the absolute configuration (1S,3S/ 1R,3S) of the synthesized compounds. The solid-state and solution ECD study of the prepared compounds, supported by ECD calculation and X-ray data, afforded a reliable ECD method for the configurational assignment of 1,3-disubstituted tetrahydro-ß-carbolines and revealed the stereochemical factors that determine the characteristic ECD data.

Loss-of-function SLC30A2 mutants are associated with gut dysbiosis and alterations in intestinal gene expression in preterm infants.

Loss of Paneth cell (PC) function is implicated in intestinal dysbiosis, mucosal inflammation, and numerous intestinal disorders, including necrotizing enterocolitis (NEC). Studies in mouse models show that zinc transporter ZnT2 (SLC30A2) is critical for PC function, playing a role in granule formation, secretion, and antimicrobial activity; however, no studies have investigated whether loss of ZnT2 function is associated with dysbiosis, mucosal inflammation, or intestinal dysfunction in humans. SLC30A2 was sequenced in healthy preterm infants (26-37 wks; n = 75), and structural analysis and functional assays determined the impact of mutations. In human stool samples, 16S rRNA sequencing and RNAseq of bacterial and human transcripts were performed. Three ZnT2 variants were common (>5%) in this population: H346Q, f = 19%; L293R, f = 7%; and a previously identified compound substitution in Exon7, f = 16%). H346Q had no effect on ZnT2 function or beta-diversity. Exon7 impaired zinc transport and was associated with a fractured gut microbiome. Analysis of microbial pathways suggested diverse effects on nutrient metabolism, glycan biosynthesis and metabolism, and drug resistance, which were associated with increased expression of host genes involved in tissue remodeling. L293R caused profound ZnT2 dysfunction and was associated with overt gut dysbiosis. Microbial pathway analysis suggested effects on nucleotide, amino acid and vitamin metabolism, which were associated with the increased expression of host genes involved in inflammation and immune response. In addition, L293R was associated with reduced weight gain in the early postnatal period. This implicates ZnT2 as a novel modulator of mucosal homeostasis in humans and suggests that genetic variants in ZnT2 may affect the risk of mucosal inflammation and intestinal disease.

Adeno-associated virus type 2 infection activates caspase dependent and independent apoptosis in multiple breast cancer lines but not in normal mammary epithelial cells.

BACKGROUND: In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery. RESULTS: AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. CONCLUSION: AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.