bluuebunny/crossref_metadata_2025 huggingface dataset:
Severe re-expansion pulmonary edema after chest tube insertion for the treatment of spontaneous pneumothorax
Authors: Song In-Hag
Published: December 2021

Rationale:

Re-expansion pulmonary edema (REPE) is a rare complication after chest tube insertion for the treatment of spontaneous pneumothorax. However, this complication can be life threatening when it occurs. Therefore, it is necessary to recognize REPE early and treat it appropriately. In the present study, we report a severe REPE case occurring after chest tube insertion in a patient with spontaneous pneumothorax.

Patient concerns:

A 27-year-old male patient visited out hospital with chest pain on the left, which had started a week ago. After diagnosed with pneumothorax and having chest tube insertion, the patient complained of sudden shortness of breath, persistent cough, foamy sputum, and vomiting.

Diagnosis:

Based on the symptoms and imaging findings, the patient was diagnosed as REPE.

Interventions:

After the condition of the patient deteriorated rapidly, he was transferred to intensive care unit and then mechanical ventilation and conservative treatment were performed after endotracheal intubation.

Outcomes:

After mechanical ventilation and conservative treatment in the intensive care unit, the symptoms and radiological findings improved, and then mechanical ventilation was weaned and the chest tube was removed from the patient. However, due to recurrent pneumothorax after removal of the chest tube, video assisted thoracoscopic surgery (VATS) wedge resection was performed. At 6 months post-operative follow up, he was well with normal radiological findings.

Lessons:

REPE occurs rarely, but once it does, it causes a serious condition that can be life-threatening. Therefore, patients with the risk factors related to it should receive a closed observation after chest tube insertion. Moreover, if REPE occurs, appropriate treatments should be carried out by recognizing it early.
Four-year follow up of macular intrachoroidal cavitation and chorioretinal atrophy
Authors: Abe Sanae , Iwase Takeshi
Published: December 2021

Rationale:

Macular intrachoroidal cavitation (ICC) is characterized by presence of a hyporeflective space beneath the unaltered retinal pigment epithelium (RPE) and is noted around the region of focal chorioretinal atrophy in eyes with pathologic myopia. The findings suggest that the patchy chorioretinal atrophy (PCA) progresses to ICC with time. However, there have been no reports describing long-term observational studies using OCT. We collected a case of PCA progression to macular ICC using OCT during a 4-year follow-up.

Patient concerns:

A 65-year-old woman presented with metamorphopsia and blurred vision in her left eye. Her best-corrected visual acuity (BCVA) was 20/20 and spherical equivalent refraction was –14.0 diopters in the left eye. Fundus examination revealed a white, well-defined PCA at the superonasal to the fovea which showed hypofluorescence determined by autofluorescence in the left eye. Sclera curved posteriorly at the superonasal to the fovea and the choroid was thickened at the area and ellipsoid zone (EZ) was disrupted in the area in OCT images. Additionally, another OCT images through the PCA showed a disappearance of the RPE–Bruch's membrane complex and a connection of blood vessels running from the sclera to the choroid.

Diagnoses:

PCA with macular ICC.

Interventions:

Observation.

Outcomes:

During 4-year follow up, the white patchy lesion and the hypofluorescence region gradually expanded. BCVA decreased with the expansion of the lesion and was 20/100 at the final visit. OCT through the fovea showed that the disorganized EZ expanded toward the ICC and the sensory retina of the fovea became thinner. Moreover, RPE–Bruch's membrane complex was not observed 3 years after the initial visit. During the follow-up period, the sensory retina was prominently displaced posteriorly to the ICC, though no obvious change was observed in the structure of the sclera.

Lessons:

In cases of PCA with macular ICC, the outer retina and RPE may initially atrophy, showing as an expansion of PCA, because the attachment between the inner retina and sclera may be weakened. This may result in the displacement of the retinal tissue into the space of macular ICC.
Radiosynthesis and biological evaluation of 18F-labeled bispecific heterodimer targeted dual gastrin-releasing peptide receptor and prostate-specific membrane antigen for prostate cancer imaging
Authors: Ye Shimin , Li Hongsheng , Hu Kongzhen , Li Li , Zhong Jiawei , Yan Qingsong , Wang Quanshi
Published: December 2021

Objective

Approximately 5% of prostatic primary tumors and 15% of metastatic tumors were found to be prostate-specific membrane antigen (PSMA)-negative. Targeting gastrin-releasing peptide receptor (GRPR) has been shown to complement patients with PSMA-negative prostate cancer (PCa). Based on previous findings, simultaneously targeting PSMA and GRPR imaging may improve the diagnosis of PCa. In this study, we report the radiosynthesis and biological evaluation of a bispecific heterodimer of NOTA-GRPR-PSMA that targeted both PSMA and GRPR for extended PCa imaging.

Methods

NOTA-GRPR-PSMA was labeled using the Al18F-chelating one-step method. The competitive combination experiment and specific binding assay were performed in vitro using 22Rv1 (PSMA+) and PC-3 (GRPR+) cells. To determine the distribution and specificity in vivo, biodistribution and micro-PET/computed tomography of [18F]AlF-GRPR-PSMA were performed on mice bearing 22Rv1 or PC-3 tumors.

Results

[18F]AlF-GRPR-PSMA had a radiochemical purity of over 98% and demonstrated high stability in vivo and in vitro, with a LogD of −1.2 ± 0.05. Cell uptake and inhibition studies of [18F]AlF-GRPR-PSMA in 22Rv1 and PC-3 cells revealed bispecific GRPR and PSMA bindings. According to the biodistribution study and PET imaging, [18F]AlF-GRPR-PSMA was mainly excreted through the kidney. Tumor uptake was high in 22Rv1 tumor (10.1 ± 0.4 %ID/g) and moderate in PC-3 tumor (2.1 ± 0.6 %ID/g) 2 h p.i., whereas blocking studies significantly decreased the tumor uptake of 22Rv1 and PC-3.

Conclusion

[18F]AlF-GRPR-PSMA has the potential to simultaneously target PSMA and GRPR for PCa imaging.
Use of methadone as an alternative to morphine for chronic pain management: a noninferiority retrospective observational study
Authors: Moreira de Barros Guilherme Antonio , Baradelli Ricardo , Rodrigues Debora Garcia , Toffoletto Odaly , Domingues Flavia Seullner , Gayoso Maisa Vitoria , Lopes Alexandre , Barros Afiune Jorge , Nunes Guimarães Gabriel Magalhães
Published: December 2021

Introduction:

Chronic pain causes disability and is prevalent in the general population. Opioids are a part of a multimodal strategy for pain management. Methadone, a cheap and long-acting synthetic opioid, may represent an option for those who have limited access to the aforementioned class of analgesics. We aimed to provide a real-world evidence for the analgesic use of methadone, compared with morphine.

Methods:

We conducted a noninferiority, retrospective observational single center study of patients with chronic pain, managed with either methadone or morphine at an outpatient specialized clinic. We extracted data from the electronic health records of patients who underwent an active treatment between August 2012 and January 2020 and were examined for at least 2 consecutive medical visits, after the administration of one of the aforementioned drugs. Data were analyzed using a generalized additive model with random-effects mixed linear method to account for the individual-related, time-related, and drug-related variations. The numeric verbal scale (0–10) was used to assess the pain severity.

Results:

From the database of 3373 patients, we included 262 patients (175 methadone and 87 morphine). In an unadjusted analysis, methadone was superior to morphine, and the mean worst pain was 0.86 points lower (95% confidence interval, −1.29 to −0.43). Moreover, methadone was superior to morphine in the adjusted analysis, with the worst pain mean being 1.24 points lower. This provided evidence for the noninferiority of methadone than morphine.

Conclusion:

Methadone was superior to morphine in a 20% noninferiority margin for reducing worst pain.
The colon microcosm: a novel in vitro model to study Candida albicans colonisation of the human colon
Authors: Lenardon Megan
Published: December 2021

Candida albicans colonises the gastro-intestinal (GI) tract of over 60% of the population. In severely ill or immune compromised patients, this fungus can escape the gut, disseminate through the body and cause systemic disease. Most research in the field has focused on defining traits that contribute directly to virulence; there are comparatively few studies which have addressed how C. albicans colonises and persists in the gut. Furthermore, such studies have typically been performed mouse models devoid of resident GI bacteria, completely neglecting the major impact of the local microbiota on GI colonisation. How, then, does C. albicans persist in the GI tract in the presence of the normal gut microbiota?

To address this question, a novel in vitro two-phase anaerobic fermentation system that simulates the human colon microenvironment has been developed. This “colon microcosm” supports the growth of human faecal microbiota in liquid anaerobic colon medium (phase 1) and C. albicans growth on agar plugs which are added to the medium to mimic the epithelial surface (phase 2). The impact of C. albicans upon the faecal microbiota is monitored by examining the planktonic phase (phase 1), whilst the effect of the microbiota on the growth of C. albicans is monitored after extracting C. albicans cells from the agar plugs (phase 2).

The results of assays carried out to validate the model will be presented, as will data from pilot studies which illustrate the potentially exploitable impact of the human GI microbiota from healthy individuals on C. albicans growth.
CRISPR-Cas9 mutagenesis and single gene reintegration suggests functional diversity within the Candida albicans TLO gene family
Authors: Fletcher Jessica , Moran Gary , Sullivan Derek
Published: December 2021

Candida albicans has between 10-15 Telomere-associated ORF family(TLO)genes, whereas its closest relative, Candida dubliniensis, has two. The Tlo proteins are components of the Mediator complex which plays an important role in transcriptional regulation. CRISPR-Cas9 mutagenesis was used to generate a TLOnull mutant of C. albicans. Phenotypic analysis of the mutant showed significantly reduced fitness, with major defects in growth rate, morphogenesis, stress resistance and virulence in a Galleria mellonellamodel. Clade representative TLOα1, TLOβ2 and TLOγ11constructs were reintroduced into the null mutant background to determine if members of the TLO gene family exhibit functional differences. The genes were reintroduced under the control of the TET1 and ENO1promoters. TLOα1and TLOβ2expression restored stress tolerance and growth rate, in some cases to the level of the WT. TLOβ2expression also showed a dramatic effect on morphology resulting in constitutive true hyphal growth. Moderate expression of TLOγ11 had no detectable effect on many of the phenotypes tested, however overexpression increased biofilm formation in Spider medium, and also conferred increased resistance to cell wall stressors. These data suggest that individual TLO genes have distinct functions and that the diversity within the TLO family may contribute to the relative success of C. albicans as a coloniser and pathogen of humans.
CO2 accelerates Candida albicans biofilm formation and reveals novel approaches to their inhibition on airway management devices
Authors: Gourlay Campbell W , Muhlschlegel Fritz A , Pentland Daniel R
Published: December 2021

C. albicans is the predominant human fungal pathogen worldwide and frequently colonises medical devices, such as voice prosthesis, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance the effects of high CO2 levels on C. albicans biofilm growth has not been investigated to date. Here, we show that 5% CO2 significantly enhances each stage of the C. albicans biofilm forming process; from attachment through maturation to dispersion, via stimulation of the Ras/cAMP/PKA signalling pathway. Transcriptome analysis of biofilm formation under elevated CO2 conditions revealed the activation of key biofilm formation pathways governed by the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown in under elevated CO2 conditions also exhibit increases in azole resistance, tolerance to nutritional immunity and enhanced glucose uptake capabilities. We thus characterise the mechanisms by which elevated CO2 promote C. albicans biofilm formation. We also investigate the possibility of re-purposing drugs that can target the CO2 activated metabolic enhancements observed in C. albicans biofilms. Using this approach we can significantly reduce multi-species biofilm formation in a high CO2 environment and demonstrate a significant extension of the lifespan of voice prostheses in a patient trial. Our research demonstrates a bench to bedside approach to tackle Candida albicans biofilm formation.
The first multi-omic interaction network in the human pathogen Candida glabrata provides novel insights into the virulence regulator, Hsp90
Authors: Crunden Julia , Haynes Ken , Diezmann Stephanie
Published: December 2021

Candida glabrata-caused candidiasis is growing but treatments remain limited by paucity of drug targets, intrinsic azole resistance and increasing resistance to other drug classes. Drug resistance is one of numerous virulence traits regulated by the chaperone, heat shock protein 90 (Hsp90) in Candida albicans via its interactions with 5% of the genome. Hsp90 also regulates key drug resistance mechanisms in C. glabrata, but little else was known about Hsp90 in this organism. Therefore, CgHsp90 interactions were elucidated by genetic and proteomic methods.

A genetic network was produced by a chemical-genetic, synthetic-sick screen on a gene-deletion library covering 16% of the genome; whilst quantitative proteomics was undertaken by tandem mass tagging on wild-type cells. Both experiments were undertaken at 30°C, 37°C and 39°C and Hsp90 was perturbed using sub-lethal concentrations of Hsp90 inhibitor.
Efforts to identify Hsp90 interactors at these host-infection associated temperatures produced a genetic network of 68 genes and a protein network of 2298 proteins. Of these, 4 genes and 261 proteins interacted with Hsp90 at all three temperatures, indicating a core Hsp90 interaction network. Intriguingly, both networks had enrichment for the “antibiotic biosynthesis” GO term. Two genes, BCY1 and MCM16, were found to interact with Hsp90 at multiple temperatures in both networks. These data indicate the divergence of Hsp90 networks between C. glabrata and its close relatives and offer important targets for further research.
Presented here is the first multi-omic interaction network in C. glabrata, focused on the virulence and drug resistance regulator, Hsp90.
In silico screening leads to novel scaffolds with both antifungal and anti-NLRP3 inflammasome activity
Authors: Lowes David , Al-waqfi Rand , Hevener Kirk , Peters Brian
Published: December 2021

Due to structural similarities that exist between established inhibitors of the NLRP3-inflammasome, sulfonylureas Glyburide and MCC-950, and herbicidal-sulfonylureas, that specifically target fungal acetohydroxyacid synthase (AHAS), we sought to determine the potential for compounds to block both inflammation and inhibit fungal growth.

In silico screening of ∼250,000 compounds was used to identify a prioritized list of chemical structures capable of inhibiting both targets. Prioritization of the top 1% of scores identified ∼70 compounds with a diverse set of scaffolds for testing in vitro. Selected hits were used to assess anti-inflammatory function in a THP-1 challenge model with LPS+ATP and resulting IC50 values were obtained. MIC and hyphal-growth assays were conducted to determine potential antifungal activity using media depleted of branched chain amino acids isoleucine and valine, to confirm on target AHAS inhibition.

Identification of hits that exhibited low micromolar activity for NLRP3 and AHAS inhibition were selected for SAR study. In vitro testing of the analogs along with molecular docking led to increased knowledge for lead optimization of the potential hits.

In silico screening has resulted in IC50 (IL-1β release) and MIC50 (fungal growth) values with low μM potency against several Candida species. In vivo validation will further confirm the potential of the scaffolds for further synthetic-modification for the rationale design of novel dual-purpose drugs
Unveiling azole resistance mechanisms in Candida glabrata clinical isolates encoding wild-type or gain-of-function CgPdr1 alleles
Authors: Salazar Sara B. , Valez Noémi , Sotti-Novais Danielle , Simões Rita , Souza José António , Faustino Maria Joana , Mendonça Catarina , Lopes Maria Manuel , Rodrigues Vitória , Mira Nuno P.
Published: December 2021

The relevance of C. glabrata as a human pathogen is linked with its poor susceptibility to azoles as well as its extreme genomic plasticity that allows the rapid acquisition of resistance. Extensive characterization of azole-resistant C. glabrata strains unveiled the central role of the transcriptional regulator CgPdr1 in the resistance phenotype, with many strains encoding hyperactive (or gain-of-function; GOF) CgPdr1 alleles. Large scale profiling of a collection of clinical C. glabrata isolates recovered in hospitals of the Lisbon area, in Portugal, led to the identification of 11 strains exhibiting resistance to fluconazole and voriconazole, while 2 were only resistant to fluconazole. Among these strains, 10 were found to encode alleles of the CgPDR1 gene harbouring multiple non-synonymous SNPs that were not found in the alleles encoded by susceptible strains, including K274Q, I392M and I803T not previously described as GOF mutations. The isolates encoding these alleles were found to over-express several CgPdr1 target genes including the azole efflux pump CgCDR1 sustaining the idea that these represent new gain-of-function CgPdr1 alleles. Only one of the identified azole-resistant strains was found to encode a CgPDR1 allele fully identical to the one encoded by susceptible strains. To better understand the resistance phenotype of this strain, its transcriptome was compared with the one of a susceptible strain and of strains encoding CgPdr1 GOF alleles. The results of this comparative transcriptomic analysis will be discussed shedding light into the different azole-resistance mechanisms evolved by C. glabrata, including those independent of CgPdr1 GOF strains.
Farnesol secretion as a possible driving force for maintaining Candida albicans as a diploid
Authors: Nickerson Kenneth , Boone Cory , Parker Kory
Published: December 2021

Candida albicans is a pathogenic dimorphic fungus which is invariably found as a diploid in patients. C. albicans secretes the sesquiterpene farnesol both as a quorum sensing molecule which blocks the yeast to hypha conversion and as a virulence factor for pathogenicity. 20-25 μM farnesol kills other competing yeasts and fungi, often by triggering apoptosis, and yet wild type diploid C. albicans tolerates 300-500 μM farnesol. The recent availability of 10 haploid strains of C. albicans (5 mating type aand 5 mating type α) allowed us to compare their production of and sensitivity to farnesol. On average, the heterozygous diploid strains of C. albicans were 2.4 times more resistant to 20-40 μM farnesol than MTLa haploid cells and 4.6 times more resistant than MTLα haploid cells. Furthermore, the MTLa haploids produce approximately 10 times more farnesol than do the MTLα haploids. Prior work concluded that haploid strains exhibited such low fitness that C. albicans was thought to be an obligate diploid. We now suggest that increased farnesol secretion by the MTLa haploids and increased farnesol sensitivity of the MTLα haploids is a mechanism for maintaining the dominant heterozygous diploid status of C. albicans. This idea is based on the observation that the a-factor peptide pheromone is farnesylated but the α-factor pheromone is not farnesylated. Our working hypothesis is that farnesol is secreted in part via Ste6 and imported in part via Ste3, the proteins which export and import the farnesylated a-pheromone. We also examined whether farnesol was excreted in extracellular vesicles.
Azole uptake in Candida auris is strongly correlated with drug resistance
Authors: Esquivel Brooke , White Theodore C.
Published: December 2021

Analyses of fluconazole uptake in clinical isolates of C. auris, with wide ranging drug resistance profiles, has revealed interesting differences within the species as well as major distinctions from other yeast species. We previously proposed that prevention of drug uptake is a potential mechanism of drug resistance and our C. auris data provide further support for this.

We developed an assay using radio-labeled fluconazole to measure intracellular azole accumulation in fungal cells. The assay is performed under glucose-replete conditions to inhibit ATP-dependent efflux. A comparative study measuring fluconazole uptake in 63 C. auris isolates as well as a panel of other species such as C. albicans, S. cerevisiae, C. glabrata, C. krusie, C. lusitanea, C. tropicalis, and C. dublinienses revealed striking C. auris phenotypes that we have not seen in other fungal species.

There is a strong correlation between fluconazole resistance and reduced drug uptake in C. auris. Fluconazole-resistant C. auris isolates had reduced levels of intracellular fluconazole accumulation compared to susceptible isolates. Drug-resistant C. auris isolates had the lowest drug accumulation of any of the yeast species tested. Fluconazole-susceptible C. auris isolates had dramatically increased fluconazole accumulation compared to the resistant isolates as well as when compared to other yeast species.

Of the 63 C. auris isolates, 28 of 32 (∼88%) resistant isolates had extremely low fluconazole uptake, whereas 15 of 18 (∼83%) susceptible isolates had high fluconazole uptake. This association between reduced drug uptake and resistance could be a C. auris-distinctive mechanism of drug resistance.
Spatial mapping of immunological epitopes in the Candida cell wall using C-type lectin probes
Authors: Vendele Ingrida , Feizi Ten , Spyrou Maria , Stappers Mark , Brown Gordon , Willment Janet , Silva Lisete , Palma Angelina , Chaie Wengang , Liu Yan , Gow Neil
Published: December 2021

The primary recognition event between a fungal pathogen and the immune system normally involves the engagement of a pattern recognition receptor with specific components of the cell wall. However, the cell wall is a complex three dimensional structure whose composition changes rapidly in accordance with environmental stimuli. Therefore it is important to know what is the precise nature of the primary recognition event, how many events occur to activate the immune response and how these recognition events are affected by changes in cell wall architecture, cellular morphogenesis and physiological adaptation of the pathogen to specific niches in the human body. We address this fundamental question using four soluble immune C-Type lectin receptor-probes which recognize specific mannans and β-1,3 glucan in the cell wall. We use these C-type lectin probes to demonstrate that mannan epitopes are differentially distributed in the inner and outer layers of fungal cell wall in a clustered or diffuse manner. Immune reactivity of fungal cell surfaces did not correlate with relatedness of different fungal species, and mannan-detecting receptor-probes discriminated between cell surface mannans generated by the same fungus growing under different conditions. These studies demonstrate that mannan-epitopes within fungal cell walls are differentially distributed and dynamically expressed as the fungus adapted to microenvironments that would be encountered in vivo.
Novel antifungal activity of Q-Griffithsin, a broad-spectrum antiviral lectin
Authors: Nabeta Henry , Kouokam Joseph C , Lasnik Amanda , Fuqua Joshua , Palmer Kenneth E
Published: December 2021

Background

There is a rising global trend in candida strains with high resistance to fluconazole and other antifungal drugs, hence the need for novel agents. Here, we investigated the anti-Candida activity of Q-Griffithsin (Q-GRFT), a lectin naturally produced by the red-sea algae, Griffithsia spp.

Methods

To assess in vitro growth inhibitory activity, C. albicans was incubated with Q-GRFT on agar plates and in broth media. We investigated GFP-bound Q-GRFT’s ability to adhere to C. albicans using fluorescence microscopy and fluorescence intensity assessments. To demonstrate in vivogrowth inhibitory activity, CBA/J mice were treated per vaginam with Q-GRFT followed by challenge with C. albicans, and fungal burden determined following vaginal lavage.

Results

Wild type fluorescently labeled Q-GRFT displayed higher fluorescence than the lectin-binding site deficient variant following incubation with C. albicans. Q-GRFT localized around the fungal cells and bound to α-mannan in the cell wall. Q-GRFT significantly inhibited C. albicans growth in broth and on agar plates, disrupted the integrity of the cell wall, and induced ROS formation. The lectin significantly inhibited the growth of C. glabrata, C. parapsilosis and C. krusei, with modest activity against C. auris CDC388 and C. auris CDC389 strains in vitro. Topical treatment resulted in a lower fungal burden compared to the vehicle control group in vaginal candidiasis.

Conclusion

Q-GRFT binds to and inhibits C. albicans growth both in vitro and in vivo. Further studies are needed to establish the mechanism of growth inhibition.
Identification and characterization of a fungal-selective glutaminyl tRNA synthetase inhibitor with potent activity against Candida albicans
Authors: Xue Alice , Veri Amanda , Zhang Xiang , Fu Ci , Lash Emma , Iyer Kali , Revie Nicole , Robbins Nicole , Boone Charles , Myers Chad , Cowen Leah
Published: December 2021

Candida albicans is the leading cause of systemic candidiasis. Effective treatment is threatened by a dearth of antifungal options and the emergence of resistance. Thus, there is an urgent need to identify novel therapeutic targets to expand our antifungal armamentarium. A promising approach is the discovery of essential genes, as most antimicrobials target essential bioprocesses. Despite detailed characterization of gene essentiality in Saccharomyces cerevisiae,defining essential targets in the pathogen of interest is necessary due to the high level of divergence between these organisms. Thus, using a machine learning algorithm we generated a comprehensive prediction of all genes essential in C. albicans. We leveraged our essentiality predictions with high-throughput screening and chemogenomic datasets to assign the mechanism of action of a previously uncharacterized compound. We identified T-035897 as a molecule with potent bioactivity against C. albicans. Prior chemogenomic profiling in S. cerevisiae suggested that T-035897 targets the glutaminyl tRNA synthetase Gln4, whose homolog in C. albicans was predicted and verified to be required for viability. To confirm the mechanism of T-035897 in C. albicans, we performed haploinsufficiency profiling,which supported Gln4as the target. In parallel, selection of resistant mutants and targeted sequencing uncovered substitutions in the Gln4 catalytic domain. Moreover, T-035897 inhibited translation in afluorescence-based reporter assay. Finally, T-035897 selectively abrogated fungal cell growth in a co-culture model with mammalian cells. Thus, we highlight the power of leveraging essentiality datasets in order to characterize compounds with potent antifungal activity in an effort to unveil novel therapeutic strategies.
A novel CRISPRi platform to study the role of essential genes in antifungal drug-resistant Candida albicans isolates
Authors: Wensing Lauren , Shapiro Rebecca , Uthayakumar Deeva , Halder Viola , Sharma Jehoshua , Proteau Yannic , Chavez Alejandro
Published: December 2021

With the emergence of antifungal resistant Candida albicans strains, the need for new antifungal drugs is critical in combating this fungal pathogen. Investigating essential genes in C. albicans is a vital step in characterizing putative antifungal drug targets. As some of these essential genes are conserved between fungal organisms, developed therapies targeting these genes have the potential to be broad range antifungals. In order to study these essential genes, classical genetic knockout or CRISPR-based approaches cannot be used as disrupting essential genes leads to lethality in the organism. Fortunately, a variation of the CRISPR system (CRISPR interference or CRISPRi) exists that enables precise transcriptional repression of the gene of interest without introducing genetic mutations. CRISPRi utilizes an endonuclease dead Cas9 protein that can be targeted to a precise location but lacks the ability to create a double-stranded break. The binding of the dCas9 protein to DNA prevents the binding of RNA polymerase to the promoter through steric hindrance thereby reducing expression. We recently published the novel use of this technology in C. albicans and are currently working on expanding this technology to large scale repression of essential genes. Through the construction of an essential gene CRISPRi-sgRNA library, we can begin to study the function of essential genes under different conditions and identify genes that are involved in critical processes such as drug tolerance in antifungal resistant background strains. These genes can ultimately be characterized as putative targets for novel antifungal drug development, or targeted as a means to sensitize drug-resistant strains to antifungal treatment.
Investigation of the interaction of keratinocytes and Candida species
Authors: Ádám Novák , Erik Zajta , Máté Csikós , Csaba Vágvölgyi , Attila Gácser
Published: December 2021

Our skin provides immunological protection against several pathogens. Skin epithelial cells respond to microbial stimuli in various ways, such as through the production of antimicrobial peptides or secretion of cytokines, although phagocytosis of potentially evading microbes was also reported.

Relatively little is known about how skin keratinocytes differentiate between the presence of pathogenic and commensal fungi. In this project, we aimed to investigate how human keratinocytes interact with different Candida species, as common colonizers of the skin. While C. albicans is a common cause of cutaneous candidiasis, C. parapsilosisis rarely associated with this disease.For the experimentshuman skin keratinocyte cell lines (HaCaT, HPV-KER)were applied andchallengedwith C. albicans (SC5314 and WO1 strains) and C. parapsilosis (GA1 and CLIB214 strains)strains.We aimedto determine the extent to which C. albicans and C. parapsilosis damage human keratinocytes, their attachment to host cells, the keratinocytes’ ability to internalize these fungi and to examinecytokine production in response to stimuli.

Our results suggest that C. albicans causes significantly more damage to human keratinocytes than C. parapsilosis and the HPV-KER cell line was more susceptibleto the infection. In both HaCaT and HPV-KER cells, the production of IL-6, IL-8, and CCL5 increased primarilyafter C. albicans infection. Based on the adhesion studies, there was a low degree of association in case of C. parapsilosis GA1 and CLIB214 compared to C. albicans SC5314 and WO1.
Extracellular RNA as a potential activator of Neutrophil Extracellular Traps by Candida albicans biofilm
Authors: Smolarz Magdalena , Zawrotniak Marcin , Satała Dorota , Rapala-Kozik Maria
Published: December 2021

Neutrophils represent the first line of innate host defense. The ability to inhibit the development of infections is associated with the involvement of several fighting strategies. The still poorly understood mechanism is netosis, involving the release of Extracellular Neutrophil Traps (NETs). NETs are complexes of chromosomal DNA and granule content. Such a web-like structure inhibits the spread of invaders.

Netosis plays a significant role in combating Candida albicans infections. It has been shown that several factors, composing C. albicans cell surface mediate NETs production. However, the development of difficult to eradicate fungal infection is associated with the formation of the biofilm structure, which partially protects the pathogen cells from contact with the host’s immune system. One of the reasons for the creation of a such protective environment is the production of the extracellular matrix (ECM). The major components of the C. albicans ECM layer are lipids, proteins, carbohydrates but also extracellular nucleic acids, among which we observed a significant RNA content.

Considering that the ECM consisting of RNA molecules is one of the first lines of contact between biofilms and neutrophils, our current studies aimed to assess the potential role of extracellular RNA in the triggering of the netosis process by human neutrophils in vitro. We showed that RNA purified from C. albicans biofilm structure and the whole cells have the capability to induction of ROS-dependent netosis pathway. Additionally, cell migration analysis indicate that RNA molecules may also be an effective chemotactic agent. This work was supported by NCN (2019/33/B/NZ6/02284).
Genetic analysis of sirtuin deacetylases in hyphal growth of Candida albicans
Authors: Zhao Guolei , Rusche Laura
Published: December 2021

Candida albicans is a major human fungal pathogen that encounters varied host environments during infection. In response to environmental cues, C. albicans switches between ovoid yeast and elongated hyphal growth forms, and this morphological plasticity contributes to virulence. Environmental changes that alter the cell’s metabolic state could be sensed by sirtuins, which are NAD+-dependent deacetylases. Here we studied the roles of three sirtuin deacetylases, Sir2, Hst1, and Hst2, in hyphal growth of C. albicans. We made single, double, and triple sirtuin knockout strains and tested their ability to switch from yeast to hyphae. We found that true hyphae formation was significantly reduced by the deletion of SIR2 but not HST1 or HST2. Moreover, the expression of hyphal-specific genes HWP1, ALS3, and ECE1 decreased in the sir2Δ/Δ mutant compared to wild-type. This regulation of hyphae formation was dependent on the deacetylase activity of Sir2, as a point mutant lacking deacetylase activity had a similar defect in hyphae formation as the sir2Δ/Δ mutant. Finally, we found that Sir2 and Hst1 were localized to the nucleus, with Sir2 specifically focused in the nucleolus. This nuclear localization suggests a role for Sir2 and Hst1 in regulating gene expression. In contrast, Hst2 was localized to the cytoplasm. In conclusion, our results suggest that Sir2 plays a critical and non-redundant role in hyphal growth of C. albicans.
Enterococcus innesii sp. nov., isolated from the wax moth Galleria mellonella
Authors: Gooch Harriet C. C. , Kiu Raymond , Rudder Steven , Baker David J. , Hall Lindsay J. , Maxwell Anthony
Published: December 2021

Four bacterial strains were isolated from two different colony sources of the wax moth Galleria mellonella. They were characterized by a polyphasic approach including 16S rRNA gene sequence analysis, core-genome analysis, average nucleotide identity (ANI) analysis, digital DNA–DNA hybridization (dDDH), determination of G+C content, screening of antibiotic resistance genes, and various phenotypic analyses. Initial analysis of 16S rRNA gene sequence identities indicated that strain GAL7T was potentially very closely related to

Enterococcus casseliflavus

and

Enterococcus gallinarum

, having 99.5–99.9 % sequence similarity. However, further analysis of whole genome sequences revealed a genome size of 3.69 Mb, DNA G+C content of 42.35 mol%, and low dDDH and ANI values between the genomes of strain GAL7T and closest phylogenetic relative

E. casseliflavus

NBRC 100478T of 59.0 and 94.5 %, respectively, indicating identification of a putative new

Enterococcus

species. In addition, all novel strains encoded the atypical vancomycin-resistance gene vanC-4. Results of phylogenomic, physiological and phenotypic characterization confirmed that strain GAL7T represented a novel species within the genus

Enterococcus

, for which the name Enterococcus innesii sp. nov. is proposed. The type strain is GAL7T (=DSM 112306T=NCTC 14608T).
Leukocyte cytokine responses in adult patients with mitochondrial DNA defects
Authors: Karan Kalpita R. , Trumpff Caroline , Cross Marissa , Englestad Kristin M. , Marsland Anna L. , McGuire Peter , Hirano Michio , Picard Martin
Published: December 2021

Patients with oxidative phosphorylation (OxPhos) defects causing mitochondrial diseases appear particularly vulnerable to infections. Although OxPhos defects modulate cytokine production in vitro and in animal models, little is known about how circulating leukocytes of patients with inherited mitochondrial DNA (mtDNA) defects respond to acute immune challenges. In a small cohort of healthy controls (n=21) and patients (n=12) with either the m.3243A>G mutation or single, large-scale mtDNA deletions, we examined: i) cytokine responses (IL-6, TNF-α, IL-1β) in response to acute lipopolysaccharide (LPS) exposure, and ii) sensitivity to the immunosuppressive effects of glucocorticoid signaling (dexamethasone) on cytokine production. In dose-response experiments to determine the half-maximal effective LPS concentration (EC50), relative to controls, leukocytes from patients with mtDNA deletions showed 74 - 79% lower responses for IL-6 and IL-1β (pIL-6=0.031, pIL-1β=0.009). Moreover, IL-6 response to LPS in presence of GC was also blunted in cells from patients with mtDNA deletions (pIL-6=0.006), but not in leukocytes from patients with the m.3243A>G mutation. Overall, these ex vivo data provide preliminary evidence that some systemic OxPhos defects may compromise immune cytokine responses and glucocorticoid sensitivity. Further work in larger cohorts is needed to define the nature of immune dysregulation in patients with mitochondrial disease, and their potential implications for disease phenotypes.
Cortical Temporal Integration Window for Binaural Cues accounts for Sluggish Auditory Spatial Perception
Authors: Singh Ravinderjit , Bharadwaj Hari M.
Published: December 2021

The auditory system has exquisite temporal coding in the periphery which is transformed into a rate-based code in central auditory structures like auditory cortex. However, the cortex is still able to synchronize, albeit at lower modulation rates, to acoustic fluctuations. The perceptual significance of this cortical synchronization is unknown. We estimated physiological synchronization limits of cortex (in humans with electroencephalography) and brainstem neurons (in chinchillas) to dynamic binaural cues using a novel system-identification technique, along with parallel perceptual measurements. We find that cortex can synchronize to dynamic binaural cues up to approximately 10 Hz, which aligns well with our measured limits of perceiving dynamic spatial information and utilizing dynamic binaural cues for spatial unmasking, i.e. measures of binaural sluggishness. We also find the tracking limit for frequency modulation (FM) is similar to the limit for spatial tracking, demonstrating that this sluggish tracking is a more general perceptual limit that can be accounted for by cortical temporal integration limits.
Lactate regulation of activation in CD8+ T cells
Authors: Barbieri Laura , Veliça Pedro , Gameiro Paulo A. , Cunha Pedro P. , Foskolou Iosifina P. , Bargiela David , Rundqvist Helene , Johnson Randall S.
Published: December 2021

CD8+ T cells infiltrate virtually every tissue to find and destroy infected or mutated cells. They often traverse varying oxygen levels and nutrient-deprived microenvironments. High glycolytic activity in tissues can result in extended exposure of cytotoxic T cells to the metabolite lactate. Lactate can be immunosuppressive, at least in part due to its association with tissue acidosis. We show here that the lactate anion is well tolerated by CD8+ T cells in pH neutral conditions. We describe how lactate is taken up by activated CD8+ T cells and is capable of displacing glucose as a carbon source. Activation in the presence of a pH neutral form of lactate significantly alters the CD8+ T cell transcriptome, including the expression of key effector differentiation markers such as granzyme B and interferon-gamma. Our studies reveal the novel metabolic features of lactate utilization by activated CD8+ T cells, and highlight the importance of lactate in shaping the differentiation and activity of cytotoxic T cells.
Comparative genomics of Acinetobacter baumannii and therapeutic bacteriophages from a patient undergoing phage therapy
Authors: Hernandez Adriana , Liu Mei , Clark James , Le Tram , Biswas Biswajit , Bishop-Lilly Kimberly A. , Henry Matthew , Quinones Javier , Hamilton Theron , Schooley Robert , Salka Scott , Young Ry , Gill Jason
Published: December 2021

In 2016, a 68-year-old patient with a disseminated multi-drug resistant Acinetobacter baumannii infection was treated using lytic bacteriophages in one of the first modern human clinical uses of phage therapy in the United States. Due to the emergency nature of the treatment there was little time to thoroughly characterize the phages used in this intervention or the pathogen itself. Here we report the genomes of the nine phages used for treatment and three strains of A. baumannii isolated prior to and during treatment. The eight phages used in the initial treatment were found to be a group of closely related T4-like myophages; the ninth phage, AbTP3Φ1, was found to be an unrelated Fri1-like podophage. Analysis of 19 A. baumannii isolates collected before and during phage treatment showed that resistance to the T4-like phages appeared as early as two days following the start of treatment. Three A. baumannii strains (TP1, TP2 and TP3) collected before and during treatment were sequenced to closure, and all contained a 3.9 Mb chromosome of sequence type 570 with a KL116 capsule locus and identical 8.7 kb plasmids. Phage-insensitive mutants of A. baumannii strain TP1 were generated in vitro and the majority of identified mutations were located in the bacterial capsule locus. The presence of the same mutation in both the in vitro mutants and in phage-insensitive isolates TP2 and TP3, which evolved in vivo during phage treatment, indicate that in vitro investigations can produce results that are relevant and predictive for the in vivo environment.
QTQTN motif upstream of the furin-cleavage site plays key role in SARS-CoV-2 infection and pathogenesis
Authors: Vu Michelle N. , Lokugamage Kumari G. , Plante Jessica A. , Scharton Dionna , Johnson Bryan A. , Sotcheff Stephanea , Swetnam Daniele M. , Schindewolf Craig , Alvarado R. Elias , Crocquet-Valdes Patricia A. , Debbink Kari , Weaver Scott C. , Walker David H. , Routh Andrew L. , Plante Kenneth S. , Menachery Vineet D.
Published: December 2021

The furin cleavage site (FCS), an unusual feature in the SARS-CoV-2 spike protein, has been spotlighted as a factor key to facilitating infection and pathogenesis by increasing spike processing 1,2. Similarly, the QTQTN motif directly upstream of the FCS is also an unusual feature for group 2B coronaviruses (CoVs). The QTQTN deletion has consistently been observed in in vitro cultured virus stocks and some clinical isolates 3. To determine whether the QTQTN motif is critical to SARS-CoV-2 replication and pathogenesis, we generated a mutant deleting the QTQTN motif (ΔQTQTN). Here we report that the QTQTN deletion attenuates viral replication in respiratory cells in vitro and attenuates disease in vivo. The deletion results in a shortened, more rigid peptide loop that contains the FCS, and is less accessible to host proteases, such as TMPRSS2. Thus, the deletion reduced the efficiency of spike processing and attenuates SARS-CoV-2 infection. Importantly, the QTQTN motif also contains residues that are glycosylated4, and disruption its glycosylation also attenuates virus replication in a TMPRSS2-dependent manner. Together, our results reveal that three aspects of the S1/S2 cleavage site – the FCS, loop length, and glycosylation – are required for efficient SARS-CoV-2 replication and pathogenesis.
An Explainable Self-Attention Deep Neural Network for Detecting Mild Cognitive Impairment Using Multi-input Digital Drawing Tasks
Authors: Ruengchaijatuporn Natthanan , Chatnuntawech Itthi , Teerapittayanon Surat , Sriswasdi Sira , Itthipuripat Sirawaj , Chotibut Thiparat , Chunharas Chaipat
Published: December 2021

Mild cognitive impairment (MCI) is an early stage of age-inappropriate cognitive decline, which could develop into dementia – an untreatable neurodegenerative disorder. An early detection of MCI is a crucial step for timely prevention and intervention. To tackle this problem, recent studies have developed deep learning models to detect MCI and various types of dementia using data obtained from the classic clock-drawing test (CDT), a popular neuropsychological screening tool that can be easily and rapidly implemented for assessing cognitive impairments in an aging population. While these models succeed at distinguishing severe forms of dementia, it is still difficult to predict the early stage of the disease using the CDT data alone. Also, the state-of-the-art deep learning techniques still face the black-box challenges, making it questionable to implement them in the clinical setting. Here, we propose a novel deep learning modeling framework that incorporates data from multiple drawing tasks including the CDT, cube-copying, and trail-making tasks obtained from a digital platform. Using self-attention and soft-label methods, our model achieves much higher classification performance at detecting MCI compared to those of a well-established convolutional neural network model. Moreover, our model can highlight features of the MCI data that considerably deviate from those of the healthy aging population, offering accurate predictions for detecting MCI along with visual explanation that aids the interpretation of the deep learning model.