Mutation of a Major Keratin Phosphorylation Site Predisposes to Hepatotoxic Injury in Transgenic Mice

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament (IF) proteins. One important physiologic function of K8/18 is to protect hepatocytes from drug-induced liver injury. Although the mechanism of this protection is unknown, marked K8/18 hyperphosphorylation occurs in association with a variety of cell stresses and during mitosis. This increase in keratin phosphorylation involves multiple sites including human K18 serine-(ser)52, which is a major K18 phosphorylation site. We studied the significance of keratin hyperphosphorylation and focused on K18 ser52 by generating transgenic mice that overexpress a human genomic K18 ser52→ ala mutant (S52A) and compared them with mice that overexpress, at similar levels, wild-type (WT) human K18. Abrogation of K18 ser52 phosphorylation did not affect filament organization after partial hepatectomy nor the ability of mouse livers to regenerate. However, exposure of S52A-expressing mice to the hepatotoxins, griseofulvin or microcystin, which are associated with K18 ser52 and other keratin phosphorylation changes, resulted in more dramatic hepatotoxicity as compared with WT K18-expressing mice. Our results demonstrate that K18 ser52 phosphorylation plays a physiologic role in protecting hepatocytes from stress-induced liver injury. Since hepatotoxins are associated with increased keratin phosphorylation at multiple sites, it is likely that unique sites aside from K18 ser52, and phosphorylation sites on other IF proteins, also participate in protection from cell stress.     

Production of D-amino acids from D,L-5-substituted hydantoins by an Agrobacterium tumefaciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity

Conversion of D,L-p-hydroxyphenylhydantoin to D-p-hydroxyphenylglycine by Agrobacterium tumefaciens RU-OR involved a racemase, an hydantoinase and an unusual D-selective N-carbamylamino acid amidohydrolase which was active at alkaline pH and was not inhibited by N-carbamyl-L-amino acids. Enzyme activity was induced by growth in media containing 2-thiouracil. A mutant strain (RU-ORL5) was isolated, which expressed both the hydantoinase and N-carbamylamino acid amidohydrolase enzymes in the absence of inducer. © Rapid Science Ltd. 1998     

Endocytosis by the corneal endothelium. I. Regulation of binding and transport of hemeproteins and peroxidase-conjugated lectins across the tissue

 Binding, internalization, and movement of hemeproteins and peroxidase-conjugated lectins across organ cultured rat corneal endothelia has been investigated. Horseradish peroxidase (HRP) type II, bound to the surface, was minimally internalized and was easily washed off. In contrast, HRP-VI bound and was rapidly internalized. Reaction product was observed in vesicles, endosomes, multivesicular bodies, and extended along the length of the intercellular space (ICS) to Descemet’s membrane. Studies at 4° C indicated HRP-VI bound uniformly along the surface in a punctate fashion. Exposure to polylysine or mannose significantly decreased uptake. Other tracers such as HRP-VIII, -IX, catalase, and microperoxidase exhibited limited uptake by the tissue. However, endothelia vigorously internalized soybean agglutinin (SBA)–HRP, and reaction product was found intracellularly and within the ICS at the cell/Descemet’s membrane interface. Internalization and the appearance of SBA–HRP within the ICS was diminished following polylysine or mannose treatment. Experiments at 4° C indicated that SBA–HRP binding and uptake were temperature sensitive. Wheat germ agglutinin (WGA)–HRP was also strongly endocytosed and reaction product was visualized within vesicles, endosomes, and multivesicular bodies. Although WGA-HRP reaction product was observed within the ICS, none was detected at the level of Descemet’s membrane. The WGA competitive sugar N-acetyl-d-glucosamine, reduced endocytosis, whereas exposure to unlabeled WGA and mannose together reduced uptake. These results indicate endothelia exhibit differential uptake of various hemeproteins and lectins which is dependent on charge, mannose receptors, and appropriate surface sugars. Accepted: 3 Mach 1998     

Survey of Physicians’ Perspectives and Knowledge about Diagnostic Tests for Bloodstream Infections

Background

Physicians rely on blood culture to diagnose bloodstream infections (BSI) despite its limitations. As new technologies emerge for rapid BSI diagnosis, optimization of their application to patient care requires an understanding of clinicians’ perspectives on BSI diagnosis and how a rapid test would influence medical decisions.

Methods

We administered a 26-question survey to practitioners in infectious diseases/microbiology, critical care, internal medicine, and hematology/oncology services in USA and Germany about current standards in diagnosing and treating BSI and a hypothetical rapid BSI test.

Results

Responses from 242 providers had roughly equal representation across specialties. For suspected BSI patients, 78% of practitioners would administer empiric broad spectrum antibiotics although they estimated, on average, that 31% of patients received incorrect antibiotics while awaiting blood culture results. The ability of blood culture to rule in or rule out infection was very/extremely acceptable in 67% and 36%, respectively. Given rapid test results, 60–87% of practitioners would narrow the spectrum of antimicrobial therapy depending on the microorganism detected, with significantly higher percentages when resistance determinants were also tested. Over half of respondents felt a rapid test would be very/extremely influential on clinical practice.

Conclusions

Limitations of blood culture were perceived as a barrier to patient care. A rapid test to diagnose BSI would impact clinical practice, but the extent of impact may be limited by prevailing attitudes and practices. Opportunities exist for interventions to influence practitioners’ behaviors in BSI management particularly with emergence of newer diagnostic tests.     

Combining the Sterile Insect Technique with Wolbachia-Based Approaches: II- A Safer Approach to Aedes albopictus Population Suppression Programmes,Designed to Minimize the Consequences of Inadvertent Female Release

Due to the absence of a perfect method for mosquito sex separation, the combination of the sterile insect technique and the incompatible insect technique is now being considered as a potentially effective method to control Aedes albopictus. In this present study first we examine the minimum pupal irradiation dose required to induce complete sterility in Wolbachia triple-infected (HC), double-infected (GUA) and uninfected (GT) female Ae. albopictus. The HC line is a candidate for Ae. albopictus population suppression programmes, but due to the risk of population replacement which characterizes this triple infected line, the individuals to be released need to be additionally irradiated. After determining the minimum irradiation dose required for complete female sterility, we test whether sterilization is sufficient to prevent invasion of the triple infection from the HC females into double-infected (GUA) populations. Our results indicate that irradiated Ae. albopictus HC, GUA and GT strain females have decreased fecundity and egg hatch rate when irradiated, inversely proportional to the dose, and the complete sterilization of females can be acquired by pupal irradiation with doses above 28 Gy. PCR-based analysis of F₁ and F₂ progeny indicate that the irradiated HC females, cannot spread the new Wolbachia wPip strain into a small cage GUA population, released at a 1:5 ratio. Considering the above results, we conclude that irradiation can be used to reduce the risk of population replacement caused by an unintentional release of Wolbachia triple-infected Ae. albopictus HC strain females during male release for population suppression.     

The “Reading the Mind in the Eyes” Test: Complete Absence of Typical Sex Difference in ~400 Men and Women with Autism

The “Reading the Mind in the Eyes” test (Eyes test) is an advanced test of theory of mind. Typical sex difference has been reported (i.e., female advantage). Individuals with autism show more difficulty than do typically developing individuals, yet it remains unclear how this is modulated by sex, as females with autism have been under-represented. Here in a large, non-male-biased sample we test for the effects of sex, diagnosis, and their interaction. The Eyes test (revised version) was administered online to 395 adults with autism (178 males, 217 females) and 320 control adults (152 males, 168 females). Two-way ANOVA showed a significant sex-by-diagnosis interaction in total correct score (F(1,711) = 5.090, p = 0.024, η_p ² = 0.007) arising from a significant sex difference between control males and females (p < 0.001, Cohen’s d = 0.47), and an absence of a sex difference between males and females with autism (p = 0.907, d = 0.01); significant case-control differences were observed across sexes, with effect sizes of d = 0.35 in males and d = 0.69 in females. Group-difference patterns fit with the extreme-male-brain (EMB) theory predictions. Eyes test-Empathy Quotient and Eyes test-Autism Spectrum Quotient correlations were significant only in females with autism (r = 0.35, r = -0.32, respectively), but not in the other 3 groups. Support vector machine (SVM) classification based on response pattern across all 36 items classified autism diagnosis with a relatively higher accuracy for females (72.2%) than males (65.8%). Nevertheless, an SVM model trained within one sex generalized equally well when applied to the other sex. Performance on the Eyes test is a sex-independent phenotypic characteristic of adults with autism, reflecting sex-common social difficulties, and provides support for the EMB theory predictions for both males and females. Performance of females with autism differed from same-sex controls more than did that of males with autism. Females with autism also showed stronger coherence between self-reported dispositional traits and Eyes test performance than all other groups.     

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.     

A comparison of populations of island and adjacent mainland species of Caribbean Selenops (Araneae: Selenopidae) spiders

The role of the landscape in structuring populations has been the focus of numerous studies, in particular, the extent to which islands provide opportunities for isolation, and the consistency of such an effect across lineages. The current study examines this phenomenon using a series of relatively widespread taxa, all within a single genus of spiders, Selenops. We focus on the Caribbean Islands and adjacent Mesoamerican mainland to examine how the islands per se dictate structure across lineages. We use molecular genetic data from mitochondrial and nuclear genes to examine the population structure of seven species of Selenops. Comparisons are made between species found in the Greater Antilles, Lesser Antilles, and adjacent mainland. Results indicate that geography has little effect on the population structure of mainland species. In contrast, population structure appears to be partitioned by island in the insular Caribbean. Within islands, the amount of population structure for each species is variable and may be dictated more by ecological or demographic parameters, rather than geographic location. The overall conclusion is that the extent to which a given lineage is structured is highly variable across species, with this variability overwhelming any general signal of geographical isolation.     

Anatomy in Toledonia warenella n. sp. (Gastropoda: Opisthobranchia: Diaphanidae) visualized by three-dimensional reconstruction

Abstract. A new species in the cephalaspidean family Diaphanidae, Toledonia warenella n. sp., is described from New South Wales, Australia. Three-dimensional, computer-aided reconstructions of the anatomy, based on serial histological sections, show that T. warenella n. sp. has some features in common with other diaphanids, including lack of jaws, gizzard plates, or an operculum. A gill was not detected in the mantle cavity, which is dominated by large, triangular, opposed ciliary bands and a kidney with lobed edges. The buccal mass is large with thick, muscular walls, perhaps indicative of a suctorial feeding behavior. Comparison with other diaphanid genera was hindered by lack of detailed published anatomical data, but shell and radula morphology suggests a close relationship between the new species and the polar genera Toledonia and Bogasonia (members of Toledoniinae), and for this reason, it is provisionally included in Toledonia. The systematics of Diaphanidae are addressed in the light of the anatomical data presented here.     

SRAP Technique Efficiently Generates Polymorphisms in Puccinia striiformis Isolates

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f.sp. tritici (PST), is a major disease of wheat in temperate‐cold climates. The identification of new markers would ease the procedure for evaluating the ongoing pathogen evolution. Twelve single pustule isolates were generated from samples of PST obtained in UK during 1987–2001. They were evaluated for their pathogenic behaviour on a set of differential cultivars and were analysed by sequence‐related amplified polymorphisms (SRAP) technique, to identify polymorphisms useful to evaluate variability among isolates. This is the first report of the application of SRAP technique to Uredinales order.