Search

Abstract One of the major hurdles that has hindered the success of chimeric antigen receptor (CAR) T cell therapies against solid tumors is on-target off-tumor (OTOT) toxicity due to sharing of the same epitopes on normal tissues. To elevate the safety profile of CAR-T cells, an affinity/avidity fine-tuned CAR was designed enabling CAR-T cell activation only in the presence of a highly expressed tumor associated antigen (TAA) but not when recognizing the same antigen at a physiological level on healthy cells. Using direct stochastic optical reconstruction microscopy (dSTORM) which provides single-molecule resolution, and flow cytometry, we identified high carbonic anhydrase IX (CAIX) density on clear cell renal cell carcinoma (ccRCC) patient samples and low-density expression on healthy bile duct tissues. A Tet-On doxycycline-inducible CAIX expressing cell line was established to mimic various CAIX densities, providing coverage from CAIX-high skrc-59 tumor cells to CAIX-low MMNK-1 cholangiocytes. Assessing the killing of CAR-T cells, we demonstrated that low-affinity/high-avidity fine-tuned G9 CAR-T has a wider therapeutic window compared to high-affinity/high-avidity G250 that was used in the first anti-CAIX CAR-T clinical trial but displayed serious OTOT effects. To assess the therapeutic effect of G9 on patient samples, we generated ccRCC patient derived organotypic tumor spheroid (PDOTS) ex vivo cultures and demonstrated that G9 CAR-T cells exhibited superior efficacy, migration and cytokine release in these miniature tumors. Moreover, in an RCC orthotopic mouse model, G9 CAR-T cells showed enhanced tumor control compared to G250. In summary, G9 has successfully mitigated OTOT side effects and in doing so has made CAIX a druggable immunotherapeutic target.

In winemaking, malolactic fermentation (MLF), which converts L-malic acid to L-lactic acid, is often applied after the alcoholic fermentation stage to improve the sensory properties of the wine and its microbiological stability. MLF is usually performed by lactic acid bacteria, which, however, are sensitive to the conditions of alcoholic fermentation. Therefore, the development of wine yeast strains capable of both alcoholic fermentation and MLF is an important task. Using genome editing, we engineered a modified variant of the triploid wine yeast strain Saccharomyces cerevisiae I-328, in which the CAR1 arginase gene was replaced by the malate permease gene from Schizosaccharomyces pombe and the malolactic enzyme gene from Oenococcus oeni. Genome-wide transcriptional profiling confirmed the expression of the introduced genes and revealed a limited effect of the modification on global gene expression. Winemaking experiments show that genome editing did not affect fermentation activity and ethanol production, while use of the modified strain resulted in a tenfold reduction in malate content with simultaneous formation of lactate. The resulting wines had a softer and more harmonious taste compared to wine obtained using the parental strain. Inactivation of arginase, which forms urea and L-ornithine through the breakdown of arginine, also resulted in a twofold decrease in the content of urea and the carcinogenic ethyl carbamate in wine. Thus, the new strain with the replacement of the arginase gene with the MLF gene cassette is promising for use in winemaking.

Abstract A combination of a high sediment input and intense bottom currents often leads to the formation of contourites (sediments deposited or significantly reworked by bottom currents). Both of these components are present in the Vema Fracture Zone valley which is the most important passageway for the distribution of the Antarctic Bottom Water from the West to the North-East of the Atlantic. However, no contourite drifts, moats or contourite channels have been found in this region in more than half a century of research. The prevailing sedimentation paradigm postulates that turbidity currents have predominantly governed sedimentation in this region during the Pleistocene. This work describes the first example of contourite depositional system identified in the Vema Fracture Zone. The discovery was made through detailed high-resolution sub-bottom profiling, as well as numerical modeling and direct measurements of bottom current velocities. Such systems are exceptionally uncommon in fracture zones. This study highlights the importance of further research of contourites along the Vema Fracture Zone based on modern concepts of contourite and mixed depositional systems. The work also emphasizes the need to reevaluate the impact of bottom currents on sedimentation in this region, and particularly in the narrow segments of the fracture zone valley.

Despite the success of KRAS G12C inhibitors in non–small cell lung cancer (NSCLC), more effective treatments are needed. One preclinical strategy has been to cotarget RAS and mTOR pathways; however, toxicity due to broad mTOR inhibition has limited its utility. Therefore, we sought to develop a more refined means of targeting cap-dependent translation and identifying the most therapeutically important eukaryotic initiation factor 4F complex–translated (eIF4F-translated) targets. Here, we show that an eIF4A inhibitor, which targets a component of eIF4F, dramatically enhances the effects of KRAS G12C inhibitors in NSCLCs and together these agents induce potent tumor regression in vivo. By screening a broad panel of eIF4F targets, we show that this cooperativity is driven by effects on BCL-2 family proteins. Moreover, because multiple BCL-2 family members are concomitantly suppressed, these agents are broadly efficacious in NSCLCs, irrespective of their dependency on MCL1, BCL-xL, or BCL-2, which is known to be heterogeneous. Finally, we show that MYC overexpression confers sensitivity to this combination because it creates a dependency on eIF4A for BCL-2 family protein expression. Together, these studies identify a promising therapeutic strategy for KRAS-mutant NSCLCs, demonstrate that BCL-2 proteins are the key mediators of the therapeutic response in this tumor type, and uncover a predictive biomarker of sensitivity.

This report presents the data on the draft genome sequence of Bifidobacterium bifidum strain ICIS-504. The strain, isolated from the intestine of a 41-year-old healthy woman is a member of community consist of four strains of three bifidobacterial species: B. longum, B. bifidum and B. breve. Annotation of the genome sequence revealed as high similarity with deposited strains as the unique duplication in the functional region of the only AmiR-family response regulator gene. The draft genome sequence data of B. bifidum strain ICIS-504 is available under the accession nos. JAJJPE000000000.1, PRJNA776132 and SAMN22746550 for NCBI Genome, Bioproject and Biosample databases, respectively.

Flor yeast strains represent a specialized group of Saccharomyces cerevisiae yeasts used for the production of sherry-like wines by biological wine aging. We sequenced the genome of the industrial flor yeast strain I-329 from a collection of microorganisms for winemaking “Magarach” and the metagenomes of two flor velums based on this strain and continuously maintained for several decades. The winery uses two processes for the production of sherry-like wine: batch aging and a continuous process similar to the criaderas–solera system. The 18S rRNA gene profiling and sequencing of metagenomes of flor velums revealed the presence of the yeasts Pichia membranifaciens and Malassezia restricta in minor amounts along with the dominant S. cerevisiae I-329 flor yeast. Bacteria Oenococcus oeni and Lentilactobacillus hilgardii together accounted for approximately 20% of the velum microbiota in the case of a batch process, but less than 1% in the velum used in the continuous process. Collection strain I-329 was triploid for all chromosomes except diploid chromosomes I and III, while the copy numbers of all chromosomes were equal in industrial velums. A comparative analysis of the genome of strain I-329 maintained in the collection and metagenomes of industrial velums revealed only several dozens of single nucleotide polymorphisms, which indicates a long-term genetic stability of this flor yeast strain under the harsh conditions of biological wine aging.

This report presents the data on the draft genome sequence of Bifidobacterium bifidum strain ICIS-202. The strain, isolated from the intestine of a young healthy woman, was deposited in the State Collection of Microorganisms of Normal Microbiota in Gabrichevsky Institute of Epidemiology and Microbiology, Moscow, Russian Federation as a prospective candidate for probiotic development. The size of the genome was 2,265,060 bp (62,4% G + C content). The annotation revealed 1771 coding sequences, including 1771 proteins, 5 rRNA, 52 tRNA, and 3 ncRNA genes. The draft genome sequence data of B. bifidum strain ICIS-202 is available in DBJ/EMBL/GenBank under the accession nos. SSMS00000000.1, PRJNA412271 and SAMN07709009 for Genome, Bioproject and Biosample databases, respectively. Keywords: WGS, Bifidobacterium, Nitrogen oxide, Cytokines, Human gut, Microbiocoenosis

FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling.

A comparative study of the concerted hydrolysis of H-, CF3-, CH3-, and Ph-substituted N-sulfinylamines (R−NSO) and isocyanates (R−NCO) was performed using B3LYP/6-31G(2d,2p). The “two-water-molecule” model was found to be sufficient for a proper description of the hydrolysis reaction for both classes of compounds. Despite their overall similar reactivity, N-sulfinylamines react across both the NS and the SO bonds, whereas isocyanates hydrolyze predominantly through the NC bond, in agreement with the proton affinities of the nitrogen and oxygen atoms. The charges on sulfur (NSO) and carbon (NCO) were found to correlate well with the activation enthalpy for hydrolysis. While the reactivity of an N-sulfinylamine toward water increases with increasing electron-withdrawing ability of the substituent, the substituent effect on isocyanates becomes notable only in the presence of the strongly electron-withdrawing CF3group. [ABSTRACT FROM AUTHOR]

In this study, we determined the levels of cytokine secretory inhibitors and the microbiota biofilms of semen from healthy and infertile subjects. A total of 118 clinical bacterial isolates were isolated and tested. Cytokine secretory inhibitors were determined based on the difference in cytokine content between the control and experimental samples of cell-free supernatants of isolated microorganisms. Biofilm formation was studied by determining the adhesion of microorganisms to the surface of a 96-well sterile plate and expressed as the optical density at 630 nm (OD630). Cell-free supernatants of Staphylococcus contained higher levels of secretory inhibitor of cytokines in conditionally healthy than in infertile patients. In contrast, in infertile men, the ability to reduce cytokine levels was more characteristic of Enterococcus and Corynebacterium. Seminal Staphylococcus, Corynebacterium, and Enterococcus isolated from infertile subjects showed a greater ability to form biofilms than the same bacteria isolated from healthy men. Further research is needed on this topic, since it is necessary to determine the relationships between decreased secretory inhibitors of cytokines, production of biofilms by bacteria in semen, and infertility. It is likely that the ability of microorganisms to change the concentration of cytokines and increase the level of biofilm formation in semen may be associated with minimal impairments of fertilizing ability, which are not detected using other methods.

The Huntington's disease (HD) CAG repeat, encoding a polymorphic glutamine tract in huntingtin, is inversely correlated with cellular energy level, with alleles over approximately 37 repeats leading to the loss of striatal neurons. This early HD neuronal specificity can be modeled by respiratory chain inhibitor 3-nitropropionic acid (3-NP) and, like 3-NP, mutant huntingtin has been proposed to directly influence the mitochondrion, via interaction or decreased PGC-1alpha expression. We have tested this hypothesis by comparing the gene expression changes due to mutant huntingtin accurately expressed in STHdh(Q111/Q111) cells with the changes produced by 3-NP treatment of wild-type striatal cells. In general, the HD mutation did not mimic 3-NP, although both produced a state of energy collapse that was mildly alleviated by the PGC-1alpha-coregulated nuclear respiratory factor 1 (Nrf-1). Moreover, unlike 3-NP, the HD CAG repeat did not significantly alter mitochondrial pathways in STHdh(Q111/Q111) cells, despite decreased Ppargc1a expression. Instead, the HD mutation enriched for processes linked to huntingtin normal function and Nf-kappaB signaling. Thus, rather than a direct impact on the mitochondrion, the polyglutamine tract may modulate some aspect of huntingtin's activity in extra-mitochondrial energy metabolism. Elucidation of this HD CAG-dependent pathway would spur efforts to achieve energy-based therapeutics in HD.