Evaluating the Antimicrobial Properties of Commercial Hand Sanitizers.

Hand sanitizers have been developed as a convenient means to decontaminate an individual's hands of bacterial pathogens in situations in which soap and water are not available. Yet to our knowledge, no study has compared the antibacterial efficacy of a large collection of hand sanitizers. Using zone of growth inhibition and kill curve assays, we assessed the performance of 46 commercially available hand sanitizers that were obtained from national chain big-box stores, gasoline stations, pharmacies, and boutiques for antibacterial activity toward prototypical Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacterial pathogens. Results revealed substantial variability in the efficacy of many sanitizers evaluated. Formulations following World Health Organization-recommended ingredients (80% ethanol or 75% isopropyl alcohol) or those including benzalkonium chloride as the active principal ingredient displayed excellent antibacterial activity, whereas others exhibited modest or poor activity in the assays performed. Results also revealed that E. coli was generally more susceptible to most sanitizers in comparison to S. aureus and that there was significant strain-to-strain variability in hand sanitizer antimicrobial efficacy regardless of the organism evaluated. Further, tests of a subset of hand sanitizers toward severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed no direct correlation between antibacterial and antiviral performance, with all ethyl alcohol formulations performing equally well and displaying improved activity in comparison to benzalkonium chloride-containing sanitizer. Taken together, these results indicate that there is likely to be substantial variability in the antimicrobial performance of commercially available hand sanitizers, particularly toward bacterial pathogens, and a need to evaluate the efficacy of sanitizers under development.IMPORTANCE In response to the coronavirus disease 2019 (COVID-19) pandemic, hand hygiene has taken on a prominent role in efforts to mitigate SARS-CoV-2 transmission and infection, which has led to a radical increase in the number and types of hand sanitizers manufactured to meet public demand. To our knowledge, no studies have evaluated or compared the antimicrobial performance of hand sanitizers that are being produced under COVID-19 emergency authorization. Tests of 46 commercially available hand sanitizers purchased from national chain brick-and-mortar stores revealed considerable variability in their antibacterial performance toward two bacterial pathogens of immediate health care concern, S. aureus and E. coli Expanded testing of a subset of hand sanitizers revealed no direct correlation between antibacterial performance of individual sanitizers and their activity toward SARS-CoV-2. These results indicate that as the pandemic subsides, there will be a need to validate the antimicrobial efficacy of sanitizers being produced.

Interaction of oxygen concentration and retention of pollutants in vertical flow constructed wetlands for CSO treatment.

The EU Water Framework Directive (WFD) calls for a good quality of all water bodies. Retention soil filters (RSF) have been developed to treat discharges from combined sewers systems. RSF have proved over the past 15 years to be the most effective measure to meet the EU WFD standards, especially for small or particularly sensitive receiving waters, which require an enhanced reduction of emissions from combined sewer overflows (CSOs). The paper presents results from laboratory-scale experiments, in which the oxygen measurement in the filter plays a main role. The results show remarkable differences in oxygen concentrations in different filter depths. The highest oxygen consumption takes place in the upper part of the filter. In the lower part the re-aeration of sewage from the soil air dominates. This indicates that the biological activity is limited to the upper part of the filter. The availability of oxygen in the filter is a sign for degradation of wastewater compounds (ammonium, COD) under certain conditions and already takes place during the filter operation. The removal of ammonium especially cannot be strictly divided into phases of sorption during the loading and oxidation during the dry period any more.

5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation.

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2'-deoxycytidine (5-aza-CdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, post-transcriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

Intersection of the Kap123p-mediated nuclear import and ribosome export pathways.

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.

The dynamics of karyopherin-mediated nuclear transport.

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.

A link between the synthesis of nucleoporins and the biogenesis of the nuclear envelope.

The nuclear pore complex (NPC) is a multicomponent structure containing a subset of proteins that bind nuclear transport factors or karyopherins and mediate their movement across the nuclear envelope. By altering the expression of a single nucleoporin gene, NUP53, we showed that the overproduction of Nup53p altered nuclear transport and had a profound effect on the structure of the nuclear membrane. Strikingly, conventional and immunoelectron microscopy analysis revealed that excess Nup53p entered the nucleus and associated with the nuclear membrane. Here, Nup53p induced the formation of intranuclear, tubular membranes that later formed flattened, double membrane lamellae structurally similar to the nuclear envelope. Like the nuclear envelope, the intranuclear double membrane lamellae enclosed a defined cisterna that was interrupted by pores but, unlike the nuclear envelope pores, they lacked NPCs. Consistent with this observation, we detected only two NPC proteins, the pore membrane proteins Pom152p and Ndc1p, in association with these membrane structures. Thus, these pores likely represent an intermediate in NPC assembly. We also demonstrated that the targeting of excess Nup53p to the NPC and its specific association with intranuclear membranes were dependent on the karyopherin Kap121p and the nucleoporin Nup170p. At the nuclear envelope, the abilities of Nup53p to associate with the membrane and drive membrane proliferation were dependent on a COOH-terminal segment containing a potential amphipathic alpha-helix. The implications of these results with regards to the biogenesis of the nuclear envelope are discussed.

Troglitazone amplifies counterregulatory responses to hypoglycemia in nondiabetic subjects.

As insulin sensitizers, thiazolidinediones could affect the hormonal counterregulatory response to hypoglycemia via the modulatory effect of insulin on counterregulation. In addition, recent studies suggest that thiazolidinediones may influence key steps in glucose sensing and glucoregulatory hormone secretion. We therefore evaluated the effects of a short course of troglitazone on counterregulatory hormones in response to mild hypoglycemia in eight lean nondiabetic subjects. Subjects received either troglitazone (400 mg/day) or placebo for 7 days before stepped hypoglycemia clamp studies (5.0, 4.4, 3.9, and 3.3 mmol/L target plasma glucose steps, 50 min each). The glycemic thresholds for secretion of epinephrine (3.77 +/- 0.05 mmol/L) and glucagon (3.83 +/- 0.11 mmol/L) were reset to a higher plasma glucose concentration after troglitazone [4.05 +/- 0.05 mmol/L (P = 0.003) and 4.10 +/- 0.05 mmol/L (P = 0.03), respectively]. In addition, the magnitude of the rise in epinephrine and glucagon concentrations was higher with troglitazone (28% and 11%, respectively; P < 0.05 for both), whereas plasma norepinephrine, GH, and cortisol were comparable in both sets of studies. Endogenous glucose production, measured with [3-(3)H]glucose, rose by 33% (P < 0.05) in the troglitazone studies compared with 17% (P = NS) after placebo. We conclude that thiazolidinediones may induce an amplification of the counterregulatory response to hypoglycemia characterized by a shift in the glycemic threshold for and an increase in the magnitude of glucagon and epinephrine secretion, and subsequent activation of glucose production.

Rrb1p, a yeast nuclear WD-repeat protein involved in the regulation of ribosome biosynthesis.

Ribosome biogenesis is regulated by environmental cues that coordinately modulate the synthesis of ribosomal components and their assembly into functional subunits. We have identified an essential yeast WD-repeat-containing protein, termed Rrb1p, that has a role in both the assembly of the 60S ribosomal subunits and the transcriptional regulation of ribosomal protein (RP) genes. Rrb1p is located in the nucleus and is concentrated in the nucleolus. Its presence is required to maintain normal cellular levels of 60S subunits, 80S ribosomes, and polyribosomes. The function of Rrb1p in ribosome biogenesis appears to be linked to its association with the ribosomal protein rpL3. Immunoprecipitation of Rrb1p from nuclear extracts revealed that it physically interacts with rpL3. Moreover, the overproduction of Rrb1p led to increases in cellular levels of free rpL3 that accumulated in the nucleus together with Rrb1p. The concentration of these proteins within the nucleus was dependent on ongoing protein translation. We also showed that overexpression of RRB1 led to an increase in the expression of RPL3 while all other examined RP genes were unaffected. In contrast, depletion of RRB1 caused an increase in the expression of all RP genes examined except RPL3. These results suggest that Rrb1p regulates RPL3 expression and uncouples it from the coordinated expression of other RP genes.

Yeast nucleoporins involved in passive nuclear envelope permeability.

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.

Transcutaneous glucose measurement using near-infrared spectroscopy during hypoglycemia.

OBJECTIVE: To analyze a transcutaneous near-infrared spectroscopy system as a technique for in vivo noninvasive blood glucose monitoring during euglycemia and hypoglycemia. RESEARCH DESIGN AND METHODS: Ten nondiabetic subjects and two patients with type 1 diabetes were examined in a total of 27 studies. In each study, the subject's plasma glucose was lowered to a hypoglycemia level (approximately 55 mg/dl) followed by recovery to a glycemic level of approximately 115 mg/dl using an intravenous infusion of insulin and 20% dextrose. Plasma glucose levels were determined at 5-min intervals by standard glucose oxidase method and simultaneously by a near-infrared spectroscopic system. The plasma glucose measured by the standard method was used to create a calibration model that could predict glucose levels from the near-infrared spectral data. The two data sets were correlated during the decline and recovery in plasma glucose, within 10 mg/dl plasma glucose ranges, and were examined using the Clarke Error Grid Analysis. RESULTS: Two sets of 1,704 plasma glucose determinations were examined. The near-infrared predictions during the fall and recovery in plasma glucose were highly correlated (r = 0.96 and 0.95, respectively). When analyzed during 10 mg/dl plasma glucose segments, the mean absolute difference between the near-infrared spectroscopy method and the chemometric reference ranged from 3.3 to 4.4 mg/dl in the nondiabetic subjects and from 2.6 to 3.8 mg/dl in the patients with type 1 diabetes. Using the Error Grid Analysis, 97.7% of all the near-infrared predictions were assigned to the A-zone. CONCLUSIONS: Our findings suggest that the near-infrared spectroscopy method can accurately predict plasma glucose levels during euglycemia and hypoglycemia in humans.

Phenotype of mast cells in the brain tumor. Capillary hemangioblastoma.

Mast cells (MC) are heterogenous cell population. In normal human brain they are not numerous. Increases in number of mast cells within CNS occur in certain disease states including neoplasms. In capillary hemangioblastoma several authors reported mast cells as a fourth cell type of the tumor. The aim of the present study was to examine phenotype and distribution of MC in cerebellar capillary hemangioblastoma by means of specific immunological markers. Study was performed on the tumor of ten affected individuals. Tumor specimens of seven cases were fixed in formalin and embedded in paraffin wax. Additional three tumours were fresh-frozen samples. Mast cells were identified with two monoclonal antibodies generated against tryptase and chymase. In all capillary hemangioblastomas mast cells were numerous exclusively in the tumor mass and only occasionally found in adjacent or far from the tumor located areas of the cerebellum. The cells contained tryptase and chymase. At periphery of hemangioblastomas some mast cells underwent degeneration and calcification. Our results confirm previous observations that mast cells are numerous in the capillary hemangioblastoma and show that most of these cells are tryptase/chymase phenotype (MCTC).

Expression of cyclooxygenase-2 in astrocytes of human brain after global ischemia.

COX-2 expression, an inducible form of cyclooxygenase was studied in human brain after ischemia-reperfusion. Previously, a prominent COX-2 upregulation was described in neurons few hours or days after ischemia but little is known about COX-2 expression in non-neuronal cells participating in post-ischemic inflammation. Aim of the study was to examine COX-2 expression in activated glial cells of individuals, who died two or more weeks after resuscitation. In the cerebral cortex of these individuals ischemic necrosis was more or less widespread. In some brain areas numerous microglial cells were found. At the centre of the necrosis high expression of COX-2 was detected in macrophages, polymorphic cells and leukocytes. At the margin of necrosis hypertrophic astrocytes were COX-2 immunopositive. Expression of COX-2 was observed also in the wall of blood vessels of necrotic brain areas and in meninges. The results of the study suggest that in the late period after global ischemia reactive and hypertrophic astrocytes and macrophages may be the major sources of prostaglandins in the human brain.

Maltoma of the thyroid in a man with Hashimoto's thyroiditis.

We report the case of a 42-yr-old man with primary thyroid lymphoma arising from mucosa-associated lymphoid tissue (MALT-lymphoma, maltoma). The patient underwent a hemithyroidectomy for a growing mass in the right lobe of the thyroid while being treated with 1-thyroxine for Hashimoto's thyroiditis. The clinical diagnosis of Hashimoto's disease was confirmed by aspiration biopsy of the mass during the course of L-thyroxine treatment. Postoperatively, histology showed atypical lymphoproliferative infiltrates suspicious of low-grade non-Hodgkin's lymphoma of mucosa-associated lymphoid tissue-type, coexisting with a reactive process typical of chronic lymphocytic thyroiditis. Immunophenotyping showed a mixed B- and T-lymphocyte population, which was nondiagnostic. However, Southern blot analysis revealed a clonal rearrangement of the Ig heavy chain gene. This case demonstrates that cytology or histology may not distinguish between reactive or low-grade lymphomatous thyroid processes. The use of molecular technique was essential to prove clonality and the presence of lymphoma.

Intonation unit analysis of conversational discourse in closed head injury.

This study employed a modification of the intonation unit analysis for conversational discourse developed by Mentis and Prutting. The percentage of total intonation units produced within separate ideational categories was calculated for groups of closed head-injured and normal control subjects as well as the examiner. No significant differences were found between subject groups or the examiner's performance within the two groups. However, significant differences were noted between the examiner's production of intonation units and the performances of both subject groups. Findings suggest the manner in which samples of conversation were elicited may have constrained the context, thereby masking potential differences between groups.

Topology and functional domains of the yeast pore membrane protein Pom152p.

Integral membrane proteins associated with the nuclear pore complex (NPC) are likely to play an important role in the biogenesis of this structure. Here we have examined the functional roles of domains of the yeast pore membrane protein Pom152p in establishing its topology and its interactions with other NPC proteins. The topology of Pom152p was evaluated by alkaline extraction, protease protection, and endoglycosidase H sensitivity assays. The results of these experiments suggest that Pom152p contains a single transmembrane segment with its N terminus (amino acid residues 1-175) extending into the nuclear pore and its C terminus (amino acid residues 196-1337) positioned in the lumen of the nuclear envelope. The functional role of these different domains was investigated in mutants that are dependent on Pom152p for viability. The requirement for Pom152p in strains containing mutations allelic to the NPC protein genes NIC96 and NUP59 could be alleviated by Pom152p's N terminus, independent of its integration into the membrane. However, complementation of a mutation in NUP170 required both the N terminus and the transmembrane segment. Furthermore, mutations in NUP188 were rescued only by full-length Pom152p, suggesting that the lumenal structures play an important role in the function of pore-side NPC structures.

Specific binding of the karyopherin Kap121p to a subunit of the nuclear pore complex containing Nup53p, Nup59p, and Nup170p.

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p-protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only beta-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.