bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of non-sedating benzodiazepines with
in vivo
antischistosomal activity
Authors: Mian Md Yeunus , Sharmin Dishary , Mondal Prithu , Belayet Jawad Bin , Hossain M. Mahmun , McCusker Paul , Ryan Kaetlyn T. , Fedorov Alexander Y. , Green Heather A. , Ericksen Spencer S. , Zamanian Mostafa , Tiruveedhula V. V. N. Phani Babu , Cook James M. , Chan John D. 
Published: August 2024

ABSTRACT

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad-spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970s. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABA
A
Rs) is not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for
in vitro
antiparasitic activity. This identified five compounds that progressed to
in vivo
screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index and point to the C3 position of the benzodiazepine ring system as a logical site for further structure–activity exploration to further optimize this chemical series.
Functional and practical insights into three lactococcal antiphage systems
Authors: Grafakou Andriana , Mosterd Cas , de Waal Paul P. , van Rijswijck Irma M. H. , van Peij Noël N. M. E. , Mahony Jennifer , van Sinderen Douwe 
Published: August 2024

ABSTRACT

The persistent challenge of phages in dairy fermentations requires the development of starter cultures with enhanced phage resistance. Recently, three plasmid-encoded lactococcal antiphage systems, named Rhea, Aristaios, and Kamadhenu, were discovered. These systems were found to confer high levels of resistance against various
Skunavirus
members. In the present study, their effectiveness against phage infection was confirmed in milk-based medium, thus validating their potential to ensure reliable dairy fermentations. We furthermore demonstrated that Rhea and Kamadhenu do not directly hinder phage genome replication, transcription, or associated translation. Conversely, Aristaios was found to interfere with phage transcription. Two of the antiphage systems are encoded on pMRC01-like conjugative plasmids, and the Kamadhenu-encoding plasmid was successfully transferred by conjugation to three lactococcal strains, each of which acquired substantially enhanced phage resistance against
Skunavirus
members. Such advances in our knowledge of the lactococcal phage resistome and the possibility of mobilizing these protective functions to bolster phage protection in sensitive strains provide practical solutions to the ongoing phage problem in industrial food fermentations.

IMPORTANCE
In the current study, we characterized and evaluated the mechanistic diversity of three recently described, plasmid-encoded lactococcal antiphage systems. These systems were found to confer high resistance against many members of the most prevalent and problematic lactococcal phage genus, rendering them of particular interest to the dairy industry, where persistent phage challenge requires the development of starter cultures with enhanced phage resistance characteristics. Our acquired knowledge highlights that enhanced understanding of lactococcal phage resistance systems and their encoding plasmids can provide rational and effective solutions to the enduring issue of phage infections in dairy fermentation facilities.
Environmentally acquired gut-associated bacteria are not critical for growth and survival in a solitary bee,
Megachile rotundata
Authors: Brar Gagandeep , Floden Madison , McFrederick Quinn , Rajamohan Arun , Yocum George , Bowsher Julia 
Published: August 2024

ABSTRACT

Social bees have been extensively studied for their gut microbial functions, but the significance of the gut microbiota in solitary bees remains less explored. Solitary bee,
Megachile rotundata
females provision their offspring with pollen from various plant species, harboring a diverse microbial community that colonizes larvae guts. The
Apilactobacillus
is the most abundant microbe, but evidence concerning the effects of
Apilactobacillus
and other provision microbes on growth and survival are lacking. We hypothesized that the presence of
Apilactobacillus
in abundance would enhance larval and prepupal development, weight, and survival, while the absence of intact microbial communities was expected to have a negative impact on bee fitness. We reared larvae on pollen provisions with naturally collected microbial communities (Natural pollen) or devoid of microbial communities (Sterile pollen). We also assessed the impact of introducing
Apilactobacillus micheneri
by adding it to both types of pollen provisions. Feeding larvae with sterile pollen +
A. micheneri
led to the highest mortality rate, followed by natural pollen +
A. micheneri
, and sterile pollen. Larval development was significantly delayed in groups fed with sterile pollen. Interestingly, larval and prepupal weights did not significantly differ across treatments compared to natural pollen-fed larvae. 16S rRNA gene sequencing found a dominance of
Sodalis
, when
A. micheneri
was introduced to natural pollen. The presence of
Sodalis
with abundant
A. michene
ri suggests potential crosstalk between both, shaping bee nutrition and health. Hence, this study highlights that the reliance on nonhost-specific environmental bacteria may not impact fitness of
M. rotundata
.

IMPORTANCE

This study investigates the impact of environmentally acquired gut microbes of solitary bee fitness with insights into the microbial ecology of bee and their health. While the symbiotic microbiome is well-studied in social bees, the role of environmental acquired microbiota in solitary bees remains unclear. Assessing this relationship in a solitary pollinator, the leaf-cutting bee,
Megachile rotundata
, we discovered that this bee species does not depend on the diverse environmental bacteria found in pollen for either its larval growth or survival. Surprisingly, high concentrations of the most abundant pollen bacteria
, Apilactobacillus micheneri
did not consistently benefit bee fitness, but caused larval mortality. Our findings also suggest an interaction between
Apilactobacillus
and the
Sodalis
and perhaps their role in bee nutrition. Hence, this study provides significant insights that contribute to understanding the fitness, conservation, and pollination ecology of other solitary bee species in the future.
Comparison of the immunogenicity of mRNA-encoded and protein HIV-1 Env–ferritin nanoparticle designs
Authors: Mu Zekun , Whitley Jill , Martik Diana , Sutherland Laura , Newman Amanda , Barr Maggie , Parks Robert , Wiehe Kevin , Cain Derek W. , Hodges Katrina Z. , Venkatayogi Sravani , Lee Esther M. , Smith Lena , Mansouri Katayoun , Edwards Robert J. , Wang Yunfei , Rountree Wes , Alameh Mohamad-Gabriel , Tam Ying , Barbosa Christopher , Tomai Mark , Lewis Mark G. , Santrai Sampa , Maughan Maureen , Tian Ming , Alt Frederick W. , Weissman Drew , Saunders Kevin O. , Haynes Barton F. 
Published: August 2024

ABSTRACT

Nucleoside-modified mRNA technology has revolutionized vaccine development with the success of mRNA COVID-19 vaccines. We used modified mRNA technology for the design of envelopes (Env) to induce HIV-1 broadly neutralizing antibodies (bnAbs). However, unlike SARS-CoV-2 neutralizing antibodies that are readily made, HIV-1 bnAb induction is disfavored by the immune system because of the rarity of bnAb B cell precursors and the cross-reactivity of bnAbs targeting certain Env epitopes with host molecules, thus requiring optimized immunogen design. The use of protein nanoparticles (NPs) has been reported to enhance B cell germinal center responses to HIV-1 Env. Here, we report our experience with the expression of Env–ferritin NPs compared with membrane-bound Env gp160 when encoded by modified mRNA. We found that well-folded Env–ferritin NPs were a minority of the protein expressed by an mRNA design and were immunogenic at 20 µg but minimally immunogenic in mice at 1 µg dose
in vivo
and were not expressed well in draining lymph nodes (LNs) following intramuscular immunization. In contrast, mRNA encoding gp160 was more immunogenic than mRNA encoding Env–NP at 1 µg dose and was expressed well in draining LN following intramuscular immunization. Thus, analysis of mRNA expression
in vitro
and immunogenicity at low doses
in vivo
are critical for the evaluation of mRNA designs for optimal immunogenicity of HIV-1 immunogens.

IMPORTANCE

An effective HIV-1 vaccine that induces protective antibody responses remains elusive. We have used mRNA technology for designs of HIV-1 immunogens in the forms of membrane-bound full-length envelope gp160 and envelope ferritin nanoparticle. Here, we demonstrated in a mouse model that the membrane-bound form induced a better response than envelope ferritin nanoparticle because of higher
in vivo
protein expression. The significance of our research is in highlighting the importance of analysis of mRNA design expression and low-dose immunogenicity studies for HIV-1 immunogens before moving to vaccine clinical trials.
The Rab6 post-Golgi secretory pathway contributes to herpes simplex virus 1 (HSV-1) egress
Authors: Bergeman Melissa H. , Velarde Kimberly , Hargis Hailee L. , Glenn Honor L. , Hogue Ian B. 
Published: August 2024

ABSTRACT

Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell’s post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the
trans
-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms.

IMPORTANCE
Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.
Probing Gag-Env dynamics at HIV-1 assembly sites using live-cell microscopy
Authors: Muecksch Frauke , Klaus Severina , Laketa Vibor , Müller Barbara , Kräusslich Hans-Georg 
Published: August 2024

ABSTRACT

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P
2
]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P
2
depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P
2
depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P
2
depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P
2
depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.

IMPORTANCE
Human immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions.
Intestinal tissue response to Shiga toxin exposure
Authors: Hanson Kendal L. , Weiss Alison Ann 
Published: August 2024

ABSTRACT

Shiga toxin (Stx) is produced by some pathogenic strains of
Escherichia coli
. To study the impact of Stx on the human intestine, we utilized human intestinal organoids and human intestinal enteroids grown as human intestinal enteroid monolayers (HIEMs) in transwells. To establish optimal experimental conditions, HIEMs were grown with or without mesenchymal cells added to the basolateral wells to recapitulate the interactions between the intestinal epithelium and the underlying mesenchyme. Monolayer barrier integrity was determined through transepithelial electrical resistance (TEER) readings. Apical saline was used on the apical surface since growth medium caused uneven development of the TEER. The medium used for epithelial cells contains added growth factors, while the mesenchymal medium lacks these growth factors. We have shown that mesenchymal cells can maintain the epithelial monolayer in the medium lacking growth factors, suggesting they produce these factors. Furthermore, growth factors produced by mesenchymal cells need to build up in the medium over time, since daily medium changes were not as effective as medium changes performed every 3 days. We have also shown that addition of growth factors is toxic to mesenchymal cells. Epithelial cells were more resistant to Stx2 than the mesenchymal cells, and mesenchymal cells contributed to epithelial cell death. Epithelial cells tolerated luminal exposure better than basolateral exposure. These studies demonstrate the importance of understanding tissue interactions in a disease state when using
in vitro
and
in vitro
models.

IMPORTANCE

These studies have cemented the need for complex cell culture models when studying host-pathogen interactions. Common animal models such as mice are resistant to
E. coli
O157:H7 infections and intestinal delivery of Stx2, while humans appear to be sensitive to both. It has been proposed that in humans, shiga toxin-producing
E. coli
-mediated intestinal damage destroys the intestinal barrier and allows basolateral access to Stx2. In mice, there is no epithelial damage; therefore, they are resistant to epithelial delivery of Stx2 while remaining sensitive to Stx2 injection. Our studies show that like mice, the human epithelial layer is quite resistant to Stx2, and it is the sensitivity of the mesenchymal cells that kills the epithelial cells. We have shown that Stx2 is transported through the intact epithelium without causing damage to the resistant epithelial layer. Understanding tissue interactions during infections is therefore critical in determining the effects of pathogens on human tissues.
Differential processing of VesB by two rhomboid proteases in
Vibrio cholerae
Authors: Roberts Cameron S. , Shannon Austin B. , Korotkov Konstantin V. , Sandkvist Maria 
Published: August 2024

ABSTRACT

Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that
Vibrio cholerae
expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the
V. cholerae
cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM.

IMPORTANCE

Despite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in
Vibrio cholerae
. This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by
V. cholerae
and other pathogenic bacteria.
Post-Devonian re-emergence and demise of stromatoporoids as major reef-builders on a Carboniferous Panthalassan seamount
Authors: Ezaki Yoichi , Masui Mitsuru , Nagai Koichi , Webb Gregory E. , Shimizu Koki , Sugama Shota , Adachi Natsuko , Sugiyama Tetsuo 
Published: August 2024

Abstract
Stromatoporoids were major reef-builders during the middle Paleozoic; however, no Carboniferous stromatoporoid reefs have been reported. The Akiyoshi Limestone Group of southwest Japan formed on a seamount in the Panthalassa Ocean from the Mississippian (Visean) to the middle Permian (Capitanian). The Early Pennsylvanian (Bashkirian) reef-core setting was well developed and laterally differentiated into several reef environments. Laminated skeletons made up of cystose or flat laminae and pillar-like vertical structures were abundant and, together with Chaetetes, contributed greatly to reef construction in most reef-core environments. The morphology of the laminated skeletons clearly indicates a stromatoporoid, probably labechiid, affinity. Thus, a lineage of reef-building stromatoporoids reappeared on a Panthalassan seamount in a locally warm-water tropical setting after the Late Devonian extinctions. Ongoing global glaciation may have resulted in enhanced ocean circulation, upwelling, and nutrient supply, especially around shallow-water seamounts, culminating in elevated carbonate saturation, which should have favored hypercalcified stromatoporoids and Chaetetes. The Chaetetes–stromatoporoid reefs remained in the Moscovian, but probably died out during the Kasimovian with intensive global cooling and frequent subaerial exposure, to be replaced by Palaeoaplysina–phylloid algal reefs. The occurrence of Bashkirian reef-building stromatoporoids indicates that Paleozoic stromatoporoids continued as reef-builders long after the Late Devonian extinction, at least in Panthalassa. This occurrence emphasizes the significance of rarely preserved open, but isolated oceanic settings like Akiyoshi for global biogeography and evolution.
Cortical inexcitability in ALS: correlating a clinical phenotype
Authors: Pavey Nathan , Hannaford Andrew , Higashihara Mana , van den Bos Mehdi , Geevasinga Nimeshan , Vucic Steve , Menon Parvathi 
Published: August 2024

BackgroundCortical inexcitability, a less studied feature of upper motor neuron (UMN) dysfunction in amyotrophic lateral sclerosis (ALS), was identified in a large cross-sectional cohort of ALS patients and their demographic and clinical characteristics were contrasted with normal or hyperexcitable ALS cohorts to assess the impact of cortical inexcitability on ALS phenotype and survival.MethodsThreshold-tracking transcranial magnetic stimulation (TMS) technique with measurement of mean short interval intracortical inhibition (SICI) differentiated ALS patients into three groups (1) inexcitable (no TMS response at maximal stimulator output in the setting of preserved lower motor neuron (LMN) function), (2) hyperexcitable (SICI≤5.5%) and (3) normal cortical excitability (SICI>5.5%). Clinical phenotyping and neurophysiological assessment of LMN function were undertaken, and survival was recorded in the entire cohort.Results417 ALS patients were recruited, of whom 26.4% exhibited cortical inexcitability. Cortical inexcitability was associated with a younger age of disease onset (p<0.05), advanced Awaji criteria (p<0.01) and Kings stage (p<0.01) scores. Additionally, patients with cortical inexcitability had higher UMN score (p<0.01), lower revised ALS Functional Rating Scale score (p<0.01) and reduced upper limb strength score (MRC UL, p<0.01). Patient survival (p=0.398) was comparable across the groups, despite lower riluzole use in the cortical inexcitability patient group (p<0.05).ConclusionThe present study established that cortical inexcitability was associated with a phenotype characterised by prominent UMN signs, greater motor and functional decline, and a younger age of onset. The present findings inform patient management and could improve patient stratification in clinical trials.