Enriched Single-Nucleus RNA-Sequencing Reveals Unique Attributes of Distal Convoluted Tubule Cells.

SIGNIFICANCE STATEMENT: High-resolution single-nucleus RNA-sequencing data indicate a clear separation between primary sites of calcium and magnesium handling within distal convoluted tubule (DCT). Both DCT1 and DCT2 express Slc12a3, but these subsegments serve distinctive functions, with more abundant magnesium-handling genes along DCT1 and more calcium-handling genes along DCT2. The data also provide insight into the plasticity of the distal nephron-collecting duct junction, formed from cells of separate embryonic origins. By focusing/changing gradients of gene expression, the DCT can morph into different physiological cell states on demand. BACKGROUND: The distal convoluted tubule (DCT) comprises two subsegments, DCT1 and DCT2, with different functional and molecular characteristics. The functional and molecular distinction between these segments, however, has been controversial. METHODS: To understand the heterogeneity within the DCT population with better clarity, we enriched for DCT nuclei by using a mouse line combining "Isolation of Nuclei Tagged in specific Cell Types" and sodium chloride cotransporter-driven inducible Cre recombinase. We sorted the fluorescently labeled DCT nuclei using Fluorescence-Activated Nucleus Sorting and performed single-nucleus transcriptomics. RESULTS: Among 25,183 DCT cells, 75% were from DCT1 and 25% were from DCT2. In addition, there was a small population (<1%) enriched in proliferation-related genes, such as Top2a , Cenpp , and Mki67 . Although both DCT1 and DCT2 expressed sodium chloride cotransporter, magnesium transport genes were predominantly expressed along DCT1, whereas calcium, electrogenic sodium, and potassium transport genes were more abundant along DCT2. The transition between these two segments was gradual, with a transitional zone in which DCT1 and DCT2 cells were interspersed. The expression of the homeobox genes by DCT cells suggests that they develop along different trajectories. CONCLUSIONS: Transcriptomic analysis of an enriched rare cell population using a genetically targeted approach clarifies the function and classification of distal cells. The DCT segment is short, can be separated into two subsegments that serve distinct functions, and is speculated to derive from different origins during development.

The crystal structure of Vibrio cholerae (6-4) photolyase reveals interactions with cofactors and a DNA-binding region.

Photolyases (PLs) reverse UV-induced DNA damage using blue light as an energy source. Of these PLs, (6-4) PLs repair (6-4)-lesioned photoproducts. We recently identified a gene from Vibrio cholerae (Vc) encoding a (6-4) PL, but structural characterization is needed to elucidate specific interactions with the chromophore cofactors. Here, we determined the crystal structure of Vc (6-4) PL at 2.5 Å resolution. Our high-resolution structure revealed that the two well-known cofactors, flavin adenine dinucleotide and the photoantenna 6,7-dimethyl 8-ribityl-lumazin (DMRL), stably interact with an α-helical and an α/ß domain, respectively. Additionally, the structure has a third cofactor with distinct electron clouds corresponding to a [4Fe-4S] cluster. Moreover, we identified that Asp106 makes a hydrogen bond with water and DMRL, which indicates further stabilization of the photoantenna DMRL within Vc (6-4) PL. Further analysis of the Vc (6-4) PL structure revealed a possible region responsible for DNA binding. The region located between residues 478 to 484 may bind the lesioned DNA, with Arg483 potentially forming a salt bridge with DNA to stabilize further the interaction of Vc (6-4) PL with its substrate. Our comparative analysis revealed that the DNA lesion could not bind to the Vc (6-4) PL in a similar fashion to the Drosophila melanogaster (Dm, (6-4)) PL without a significant conformational change of the protein. The 23rd helix of the bacterial (6-4) PLs seems to have remarkable plasticity, and conformational changes facilitate DNA binding. In conclusion, our structure provides further insight into DNA repair by a (6-4) PL containing three cofactors.

Immunosuppressive calcineurin inhibitor cyclosporine A induces proapoptotic endoplasmic reticulum stress in renal tubular cells.

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA-like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA-like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA.

Ketoconazole-loading strategy to improve antifungal activity and overcome cytotoxicity on human renal proximal tubular epithelial cells.

Ketoconazole (KTZ), an antifungal agent used to treat localized or systemic fungal infections by inhibiting ergosterol synthesis, exhibits restricted efficacy within eukaryotic cells owing to its elevated toxicity and limited solubility in water. This study aims to improve the biological activity and overcome cytotoxic effects in the renal system of the hydrophobic KTZ by incorporating it into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) utilizing biomaterial nano-engineering techniques. KTZ-loaded PLGA NPs (KTZ-NPs) were prepared by single emulsion solvent evaporation method and characterized by using dynamic light scattering (DLS), electrophoretic light scattering (ELS), Fourier transform-infrared (FT-IR) spectroscopy and scanning light microscopy (SEM). Particle size and zeta potential of KTZ-NPs were determined as 182.0 ± 3.27 nm and -27.4 ± 0.56 mV, respectively. Antifungal activity was analyzed with the time-kill and top agar dilution methods onCandida albicans(C. albicans) andAspergillus flavus(A. flavus). Both KTZ and KTZ-NPs caused a significant decrease inA. flavuscell growth; however, the same effect was only observed in time-killing analysis onC. albicans, indicating a methodological difference in the antifungal analysis. According to the top agar method, the MIC value of KTZ-NPs againstA. flavuswas 9.1µg ml-1, while the minimum inhibition concentration (MIC) value of KTZ was 18.2µg ml-1. The twofold increased antifungal activity indicates that nanoparticular drug delivery systems enhance the water solubility of hydrophobic drugs. In addition, KTZ-NPs were not cytotoxic on human renal proximal tubular epithelial cells (HRPTEpCs) at fungistatic concentration, thus reducing fungal colonization without cytotoxic on renal excretion system cells.

The neutralization effect of montelukast on SARS-CoV-2 is shown by multiscale in silico simulations and combined in vitro studies.

Small molecule inhibitors have previously been investigated in different studies as possible therapeutics in the treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In the current drug repurposing study, we identified the leukotriene (D4) receptor antagonist montelukast as a novel agent that simultaneously targets two important drug targets of SARS-CoV-2. We initially demonstrated the dual inhibition profile of montelukast through multiscale molecular modeling studies. Next, we characterized its effect on both targets by different in vitro experiments including the enzyme (main protease) inhibition-based assay, surface plasmon resonance (SPR) spectroscopy, pseudovirus neutralization on HEK293T/hACE2+TMPRSS2, and virus neutralization assay using xCELLigence MP real-time cell analyzer. Our integrated in silico and in vitro results confirmed the dual potential effect of montelukast both on the main protease enzyme inhibition and virus entry into the host cell (spike/ACE2). The virus neutralization assay results showed that SARS-CoV-2 virus activity was delayed with montelukast for 20 h on the infected cells. The rapid use of new small molecules in the pandemic is very important today. Montelukast, whose pharmacokinetic and pharmacodynamic properties are very well characterized and has been widely used in the treatment of asthma since 1998, should urgently be completed in clinical phase studies and, if its effect is proved in clinical phase studies, it should be used against coronavirus disease 2019 (COVID-19).

Data management in the digital health environment scale development study.

PURPOSE: This study aims to develop a scale that measures individuals' perceptions of privacy, security, use, sharing, benefit and satisfaction in the digital health environment. METHOD: Within the scope of the study, in the scale development process; The stages of literature review, creation of items, getting expert opinion, conducting a pilot study, ensuring construct and criterion validity, and reliability analyses were carried out. The literature was searched for the formation of the question items. To evaluate the created question items, expert opinion was taken, and the question items were arranged according to the feedback from the experts. In line with the study's purpose and objectives, the focus group consisted of individuals aged 18 and above within the community. The convenience sampling method was employed for sample selection. Data were collected using an online survey conducted through Google Forms. Before commencing the survey, participants were briefed on the research's content. A pilot study was conducted with 30 participants, and as a result of the feedback from the participants, eliminations were made in the question items and the scale was made ready for application. The research was conducted by reference to 812 participants in the community. Expert evaluations of the question items were obtained, and a pilot study was conducted. A sociodemographic information form, a scale developed by the researcher, Norman and Skinner's e-Health Literacy Scale, and the Mobile Health and Personal Health Record Management Scale were used as data collection tools. RESULTS: The content validity of the research was carried out by taking expert opinions and conducting a pilot study. Exploratory factor analysis and confirmatory factor analysis were performed to ensure construct validity. The total variance explained by the scale was 60.43%. The results of confirmatory factor analysis indicated that the 20-Item 5-factor structure exhibited good fit values. According to the analysis of criterion validity, there are significant positive correlations among the Data Management in the Digital Health Environment Scale, Norman and Skinner's e-Health Literacy Scale and the Mobile Health and Personal Health Record Management Scale (p < 0.01; r = .669, .378). The Cronbach's alpha value of the scale is .856, and the test-retest reliability coefficient is .909. CONCLUSION: The Data Management in the Digital Health Environment Scale is a valid and reliable measurement tool that measures individuals' perceptions of privacy, security, use, sharing, benefit and satisfaction in the digital health environment.

Bridging the Gap between Gut Microbiota and Alzheimer's Disease: A Metaproteomic Approach for Biomarker Discovery in Transgenic Mice.

Alzheimer's Disease (AD) is a progressively debilitating form of dementia that affects millions of individuals worldwide. Although a vast amount of research has investigated the complex interplay between gut microbiota and neurodegeneration, the metaproteomic effects of microbiota on AD pathogenesis remain largely uncharted territory. This study aims to reveal the role of gut microbiota in AD pathogenesis, particularly regarding changes in the proteome and molecular pathways that are intricately linked to disease progression. We operated state-of-the-art Nano-Liquid Chromatography Mass Spectrometry (nLC-MS/MS) to compare the metaproteomic shifts of 3-month-old transgenic (3M-ALZ) and control (3M-ALM, Alzheimer's Littermate) mice, depicting the early onset of AD with those of 12-month-old ALZ and ALM mice displaying the late stage of AD. Combined with computational analysis, the outcomes of the gut-brain axis-focused inquiry furnish priceless knowledge regarding the intersection of gut microbiota and AD. Accordingly, our data indicate that the microbiota, proteome, and molecular changes in the intestine arise long before the manifestation of disease symptoms. Moreover, disparities exist between the normal-aged flora and the gut microbiota of late-stage AD mice, underscoring that the identified vital phyla, proteins, and pathways hold immense potential as markers for the early and late stages of AD. Our research endeavors to offer a comprehensive inquiry into the intricate interplay between gut microbiota and Alzheimer's Disease utilizing metaproteomic approaches, which have not been widely adopted in this domain. This highlights the exigency for further scientific exploration to elucidate the underlying mechanisms that govern this complex and multifaceted linkage.

Comparison of decision-making under ambiguity in patients with temporal lobe and frontal lobe epilepsy.

OBJECTIVE: The effect of the frontal lobe on cognitive functions is a subject that has been studied frequently. However, cognitive impairments that can be seen in frontal lobe epilepsy are less addressed. In previous studies on decision-making disorders in patients with epilepsy, patients with temporal lobe epilepsy (TLE) were frequently studied, and it was reported that decision-making disorders could be encountered in this patient group. In this study, we aimed to compare the decision-making performance of patients with cryptogenic frontal lobe epilepsy (FLE) and TLE in ambiguous situations. METHODS: Twenty patients with TLE (mean age: 34.10 ±â€¯11.71 years) and 20 patients with FLE (mean age: 32.25 ±â€¯11.92 years) were enrolled in the study and their cognitive performance was compared with 20 healthy controls (mean age: 33.15 ±â€¯13.66 years). Neuropsychological tests were performed on the participants for sleep, depression, anxiety, impulsivity, intelligence, attention, language functions, memory and learning, and frontal axis functions. Decision-making performance in ambiguous situations was studied using the Iowa Gambling Task (IGT). RESULTS: Iowa Gambling Task performances of patients with FLE and TLE were found to be worse than in healthy controls (p = 0.049). Although there was no statistically significant difference when the decision-making of patients with TLE and FLE was compared, it was observed that patients with FLE chose higher risk cards compared with those with TLE. The performances of the neuropsychological subgroup tests of patients with TLE and FLE in attention, language functions, memory and learning, and frontal axis functions were found to be significantly worse than in healthy subjects. CONCLUSION: Decision-making in patients with TLE and FLE in ambiguous situations is similarly impaired compared with healthy controls.

Four amino acids define the CO2 binding pocket of enoyl-CoA carboxylases/reductases.

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2 molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2 within the active site of Ecr from Kitasatospora setae Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2 molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2 fixation. Altogether, our study reveals unprecedented molecular details of selective CO2 binding and C-C-bond formation during the catalytic cycle of nature's most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2 in biology and chemistry.

Comprehensive Research on Past and Future Therapeutic Strategies Devoted to Treatment of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly debilitating fatal neurodegenerative disorder, causing muscle atrophy and weakness, which leads to paralysis and eventual death. ALS has a multifaceted nature affected by many pathological mechanisms, including oxidative stress (also via protein aggregation), mitochondrial dysfunction, glutamate-induced excitotoxicity, apoptosis, neuroinflammation, axonal degeneration, skeletal muscle deterioration and viruses. This complexity is a major obstacle in defeating ALS. At present, riluzole and edaravone are the only drugs that have passed clinical trials for the treatment of ALS, notwithstanding that they showed modest benefits in a limited population of ALS. A dextromethorphan hydrobromide and quinidine sulfate combination was also approved to treat pseudobulbar affect (PBA) in the course of ALS. Globally, there is a struggle to prevent or alleviate the symptoms of this neurodegenerative disease, including implementation of antisense oligonucleotides (ASOs), induced pluripotent stem cells (iPSCs), CRISPR-9/Cas technique, non-invasive brain stimulation (NIBS) or ALS-on-a-chip technology. Additionally, researchers have synthesized and screened new compounds to be effective in ALS beyond the drug repurposing strategy. Despite all these efforts, ALS treatment is largely limited to palliative care, and there is a strong need for new therapeutics to be developed. This review focuses on and discusses which therapeutic strategies have been followed so far and what can be done in the future for the treatment of ALS.

In Vitro and In Silico Study of Analogs of Plant Product Plastoquinone to Be Effective in Colorectal Cancer Treatment.

Plants have paved the way for the attainment of molecules with a wide-range of biological activities. However, plant products occasionally show low biological activities and/or poor pharmacokinetic properties. In that case, development of their derivatives as drugs from the plant world has been actively performed. As plant products, plastoquinones (PQs) have been of high importance in anticancer drug design and discovery; we have previously evaluated and reported the potential cytotoxic effects of a series of PQ analogs. Among these analogs, PQ2, PQ3 and PQ10 were selected for National Cancer Institute (NCI) for in vitro screening of anticancer activity against a wide range of cancer cell lines. The apparent superior anticancer potency of PQ2 on the HCT-116 colorectal cancer cell line than that of PQ3 and PQ10 compared to other tested cell lines has encouraged us to perform further mechanistic studies to enlighten the mode of anti-colorectal cancer action of PQ2. For this purpose, its apoptotic effects on the HCT-116 cell line, DNA binding capacity and several crucial pharmacokinetic properties were investigated. Initially, MTT assay was conducted for PQ2 at different concentrations against HCT-116 cells. Results indicated that PQ2 exhibited significant cytotoxicity in HCT-116 cells with an IC50 value of 4.97 ± 1.93 µM compared to cisplatin (IC50 = 26.65 ± 7.85 µM). Moreover, apoptotic effects of PQ2 on HCT-116 cells were investigated by the annexin V/ethidium homodimer III staining method and PQ2 significantly induced apoptosis in HCT-116 cells compared to cisplatin. Based on the potent DNA cleavage capacity of PQ2, molecular docking studies were conducted in the minor groove of the double helix of DNA and PQ2 presented a key hydrogen bonding through its methoxy moiety. Overall, both in vitro and in silico studies indicated that effective, orally bioavailable drug-like PQ2 attracted attention for colorectal cancer treatment. The most important point to emerge from this study is that appropriate derivatization of a plant product leads to unique biologically active compounds.

An Exploratory Study of Cognitive Involvement in Hereditary Transthyretin Amyloidosis.

OBJECTIVES: Hereditary amyloidogenic transthyretin (ATTRv) amyloidosis is an autosomal dominant disorder caused by mutations of the transthyretin (TTR) gene. The mutant ATTRv protein causes a systemic accumulation of amyloid fibrils in various organs. TTR is an important protein in the central nervous system physiology for the maintenance of normal cognitive process during aging, amidated neuropeptide processing, and nerve regeneration. The neuroprotective effect of transthyretin has been widely documented in animal models. Cognitive consequences of the mutant TTR in hereditary ATTRv amyloidosis patients remain still to be elucidated. We designed this study to investigate the cognitive involvement in ATTRv amyloidosis. METHODS: Detailed neuropsychological tests and cranial MRIs were performed. Biomarkers including amyloid beta 1-42, total tau, and phosphorylated tau were investigated in the cerebrospinal fluid samples. RESULTS: Median age of the cohort was 52 years (ranges 34-72). Neuropsychological assessment results were compatible with impaired executive functions (in all patients except one with only bilateral carpal tunnel syndrome, long-term visual and long-term verbal memory (severe in four patients and moderate in one). Visuospatial judgment and perception were impaired in six. Mean cerebrospinal fluid Aß1-42 (pg/ml) was 878.0 ± 249.5 in patients with cortical atrophyin MRI whereas 1210.0 ± 45.9 in patients without any cortical atrophy. Cranial MRI showed cortical atrophy in six patients (6/10). CONCLUSION: Our data showed the significance of the TTR protein in cognitive functions and highlighted the importance of the close follow-up of cognitive functions in ATTRv amyloidosis patients.

Microbiota profiling and screening of the lipase active halotolerant yeasts of the olive brine.

Searching for novel enzymes that could be active in organic solvents has become an area of interest in recent years. Olive brine naturally provides a suitable environment for the survival of halophilic and acidophilic microorganisms and the resulting genome is thought to be a gene source for determining the halophilic and acidophilic proteins that are active in a non-aqueous organic solvent medium, and so it has been used in several biotechnological and industrial applications. In this study, microbial analysis of natural, cracked green olive brine from the southern region of Turkey has been made by next-generation sequencing of the brine metagenome for the first time in the literature. The number of reads assigned to fungal operational taxonomic units was the highest percentage (73.04%) with the dominant representation of Ascomycota phylum (99% of fungi). Bacterial OTU was 3.56% of the reads and Proteobacteria phylum was 65% of the reads. The lipase production capacity of the yeasts that were grown on the media containing elevated concentrations of NaCl (1-3 M) was determined on a Rhodamine B-including medium. Molecular identification of the selected yeasts was performed and 90% of sequenced yeasts had a high level of similarity with Candida diddensiae, whereas 10% showed similarity to Candida boidinii. The hydrolytic lipase activities using olive oil were analyzed and both yeasts showed cell-bound lipase activity at pH 3.0.

Structure of the 30S ribosomal decoding complex at ambient temperature.

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.

Cognition of the mothers of patients with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) has been found to be associated with cognitive impairment. However, few studies have addressed cognitive impairment among mothers of children with DMD. In the present study, the neuropsychological profiles of both carrier mothers (C-Ms) and noncarrier mothers (NC-Ms) were examined, and the findings were compared with healthy control mothers (HC-Ms). There were 90 participants, consisting of 31 C-Ms, 24 NC-Ms, and 35 HC-Ms, each of whom completed a neuropsychological test battery. C-Ms had poorer cognition performance in attention, working memory, immediate verbal memory, visuospatial skills, and executive functions than NC-Ms, and HC-Ms. This study provides evidence that there may be cognitive impairment in mothers of patients with DMD. The cognitive impairment of C-Ms has similarities to that seen in children with DMD.

Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature.

The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II.

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source.

The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world's first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

Low-signal limit of X-ray single particle diffractive imaging.

An outstanding question in X-ray single particle imaging experiments has been the feasibility of imaging sub 10-nm-sized biomolecules under realistic experimental conditions where very few photons are expected to be measured in a single snapshot and instrument background may be significant relative to particle scattering. While analyses of simulated data have shown that the determination of an average image should be feasible using Bayesian methods such as the EMC algorithm, this has yet to be demonstrated using experimental data containing realistic non-isotropic instrument background, sample variability and other experimental factors. In this work, we show that the orientation and phase retrieval steps work at photon counts diluted to the signal levels one expects from smaller molecules or with weaker pulses, using data from experimental measurements of 60-nm PR772 viruses. Even when the signal is reduced to a fraction as little as 1/256, the virus electron density determined using ab initio phasing is of almost the same quality as the high-signal data. However, we are still limited by the total number of patterns collected, which may soon be mitigated by the advent of high repetition-rate sources like the European XFEL and LCLS-II.

Serial Femtosecond X-Ray Diffraction of HIV-1 Gag MA-IP6 Microcrystals at Ambient Temperature.

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cell's plasma membrane. High-resolution structures of the MA domain determined using cryo X-ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first X-ray free-electron laser (XFEL) study of the HIV-1 MA domain's interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using an XFEL to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of the MA-IP6 complex both diffracted to beyond 3.5 Å resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and will provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.