Chlamydia trachomatispossesses a cryptic 7.5-kb plasmid of unknown function. Here, we describe a comprehensive molecular and biological characterization of the naturally occurring plasmidless humanC. trachomatisstrain L2(25667R). We found that despite minimal chromosomal polymorphisms, the LGV strain L2(25667R) was indistinguishable from plasmid-positive strain L2(434) with regard to its in vitro infectivity characteristics such as growth kinetics, plaquing efficiency, and plaque size. The only in vitro phenotypic differences between L2(434) and L2(25667R) were the accumulation of glycogen granules in the inclusion matrix and the lack of the typical intrainclusion Brownian-like movement characteristic ofC. trachomatisstrains. Conversely, we observed a marked difference between the two strains in their abilities to colonize and infect the female mouse genital tract. The 50% infective dose of plasmidless strain L2(25667R) was 400-fold greater (4 × 10⁶inclusion-forming units [IFU]) than that of plasmid-bearing strain L2(434) (1 × 10⁴IFU). Transcriptome analysis of the two strains demonstrated a decrease in the transcript levels of a subset of chromosomal genes for strain L2(25667R). Among those genes wasglgA, encoding glycogen synthase, a finding consistent with the failure of L2(25667R) to accumulate glycogen granules. These findings support a primary role for the plasmid in in vivo infectivity and suggest that virulence is controlled, at least in part, by the plasmid's ability to regulate the expression of chromosomal genes. Our findings have important implications in understanding a role for the plasmid in the pathogenesis of human infection and disease.

Despite increasing associated mortality and morbidity, the diagnosis of fungal infections, especially with Aspergillus fumigatus (A. fumigatus), remains challenging. Based on known ability of Aspergillus species to utilize sorbitol, we evaluated 2-[¹⁸F]-fluorodeoxysorbitol (FDS), a recently described Enterobacterales imaging ligand, in animal models of A. fumigatus infection, in comparison with 2-[¹⁸F]-fluorodeoxyglucose (FDG). In vitro assays showed slightly higher ³H-sorbitol uptake by live compared with heat-killed A. fumigatus. However, this was 10.6-fold lower than E. coli uptake. FDS positron emission tomography (PET) imaging of A. fumigatus pneumonia showed low uptake in infected lungs compared with FDG (0.290 ± 0.030 vs. 8.416 ± 0.964 %ID/mL). This uptake was higher than controls (0.098 ± 0.008 %ID/mL) and minimally higher than lung inflammation (0.167 ± 0.007 %ID/mL). In the myositis models, FDS uptake was highest in live E. coli infections. Uptake was low in A. fumigatus myositis model and only slightly higher in live compared with the heat-killed side. In conclusion, we found low uptake of ³H-sorbitol and FDS by A. fumigatus cultures and infection models compared with E. coli, likely due to the need for induction of sorbitol dehydrogenase by sorbitol. Our findings do not support FDS as an Aspergillus imaging agent. At this point, FDS remains more selective for imaging Gram-negative Enterobacterales.

Background: HIV-1–associated neurocognitive disorders (HAND) progression is related to continued inflammation despite undetectable viral loads and may be caused by early viral proteins expressed by latently infected cells. Astrocytes represent an HIV reservoir in the brain where the early viral neurotoxin negative factor (Nef) is produced. We previously demonstrated that astrocytic expression of Nef in the hippocampus of rats causes inflammation, macrophage infiltration, and memory impairment. Since these processes are affected by TGFβ signaling pathways, and TGFβ-1 is found at higher levels in the central nervous system of HIV-1+ individuals and is released by astrocytes, we hypothesized a role for TGFβ-1 in our model of Nef neurotoxicity.Methods: To test this hypothesis, we compared cytokine gene expression by cultured astrocytes expressing Nef or green fluorescent protein. To determine the role of Nef and a TGFβRI inhibitor on memory and learning, we infused astrocytes expressing Nef into the hippocampus of rats and then treated them daily with an oral dose of SD208 (10 mg/kg) or placebo for 7 days. During this time, locomotor activity was recorded in an open field and spatial learning tested in the novel location recognition paradigm. Postmortem tissue analyses of inflammatory and signaling molecules were conducted using immunohistochemistry and immunofluorescence.Results: TGFβ-1 was induced in cultures expressing Nef at 24 h followed by CCL2 induction which was prevented by blocking TGFβRI with SD208 (competitive inhibitor). Interestingly, Nef seems to change the TGFβRI localization as suggested by the distribution of the immunoreactivity. Nef caused a deficit in spatial learning that was recovered upon co-administration of SD208. Brain tissue from Nef-treated rats given SD208 showed reduced CCL2, phospho-SMAD2, cluster of differentiation 163 (CD163), and GFAP immunoreactivity compared to the placebo group.Conclusions: Consistent with our previous findings, rats treated with Nef showed deficits in spatial learning and memory in the novel location recognition task. In contrast, rats treated with Nef + SD208 showed better spatial learning suggesting that Nef disrupts memory formation in a TGFβ-1-dependent manner. The TGFβRI inhibitor further reduced the induction of inflammation by Nef which was concomitant with decreased TGFβ signaling. Our findings suggest that TGFβ-1 signaling is an intriguing target to reduce neuroHIV.

The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus -specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[ ¹⁸ F]fluorocellobiose ([ ¹⁸ F]FCB) by enzymatic conversion of 2-deoxy-2-[ ¹⁸ F]fluoroglucose ([ ¹⁸ F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [ ¹⁸ F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus , bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of β-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [ ¹⁸ F]FCB into glucose and [ ¹⁸ F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [ ¹⁸ F]FCB is a promising and clinically translatable Aspergillus -specific PET tracer.

Radiolabeled antibodies against the HIV-1 envelope protein, gp120, have been previously tested in animal models and in people with HIV (PWH). Nanobodies offer advantages over antibodies, including smaller size and faster clearance, which allow labeling with fluorine-18. In this study, three nanobodies (J3, 3E3, B9) chosen based on their binding properties to the conserved CD4-binding site of gp120 were labeled with fluorine-18 and used for PET imaging in mice bearing wild-type (WT) and/or gp120-expressing (Env+) tumors. [¹⁸F]J3 and [¹⁸F]3E3 selectively targeted Env+ tumors and not WT tumors, with minimal background signal. Switching from non-site-specific radiolabeling method to sortase A-mediated site-specific conjugation at the C-terminus improved binding to Env+ tumors for all nanobodies. Site-specifically ¹⁸F-labeled J3 nanobody is the most promising candidate with the highest level of binding. These results establish an Env+ imaging method that will enable next stage testing in an HIV-1 preclinical infection model and potentially in PWH.

The science, technology, engineering, and mathematics (STEM) fields in the United States are currently facing a crisis: graduate programs are not adequately preparing all trainees for the diverse career paths on which they embark. Over recent years, this problem has intensified as the number of Ph.D. holders increased and academic research jobs stagnated or shrunk. Still, most STEM doctoral programs have maintained the singular focus on training students for academic careers at a cost to the individual’s career, society, and the economy. Universities and graduate institutions must adapt to meet the increasing demand for STEM laborers in non-academic sectors and provide relevant and robust training to their students. We propose amending the STEM Opportunities Act of 2019 to incentivize institutions to integrate experiential learning and expand training services. Provided diverse career-training, highly-skilled Ph.D. graduates will more efficiently enter and fill the STEM workforce, stimulating the U.S. economy. Addressed to: The Committee on Science, Space, and Technology, United States House of Representatives; and the Senate Committee on Health, Education, Labor, and Pensions.