High-fidelity intravoxel incoherent motion parameter mapping using locally low-rank and subspace modeling.

PURPOSE: Intravoxel incoherent motion (IVIM) is a quantitative magnetic resonance imaging (MRI) method used to quantify perfusion properties of tissue non-invasively without contrast. However, clinical applications are limited by unreliable parameter estimates, particularly for the perfusion fraction (f) and pseudodiffusion coefficient (D*). This study aims to develop a high-fidelity reconstruction for reliable estimation of IVIM parameters. The proposed method is versatile and amenable to various acquisition schemes and fitting methods. METHODS: To address current challenges with IVIM, we adapted several advanced reconstruction techniques. We used a low-rank approximation of IVIM images and temporal subspace modeling to constrain the magnetization dynamics of the bi-exponential diffusion signal decay. In addition, motion-induced phase variations were corrected between diffusion directions and b-values, facilitating the use of high SNR real-valued diffusion data. The proposed method was evaluated in simulations and in vivo brain acquisitions in six healthy subjects and six individuals with a history of SARS-CoV-2 infection and compared with the conventionally reconstructed magnitude data. Following reconstruction, IVIM parameters were estimated voxel-wise. RESULTS: Our proposed method reduced noise contamination in simulations, resulting in a 60%, 58.9%, and 83.9% reduction in the NRMSE for D, f, and D*, respectively, compared to the conventional reconstruction. In vivo, anisotropic properties of D, f, and D* were preserved with the proposed method, highlighting microvascular differences in gray matter between individuals with a history of COVID-19 and those without (p = 0.0210), which wasn't observed with the conventional reconstruction. CONCLUSION: The proposed method yielded a more reliable estimation of IVIM parameters with less noise than the conventional reconstruction. Further, the proposed method preserved anisotropic properties of IVIM parameter estimates and demonstrated differences in microvascular perfusion in COVID-affected subjects, which weren't observed with conventional reconstruction methods.

Human trypanolytic factor APOL1 forms pH-gated cation-selective channels in planar lipid bilayers: relevance to trypanosome lysis.

Apolipoprotein L-1 (APOL1), the trypanolytic factor of human serum, can lyse several African trypanosome species including Trypanosoma brucei brucei, but not the human-infective pathogens T. brucei rhodesiense and T. brucei gambiense, which are resistant to lysis by human serum. Lysis follows the uptake of APOL1 into acidic endosomes and is apparently caused by colloid-osmotic swelling due to an increased ion permeability of the plasma membrane. Here we demonstrate that nanogram quantities of full-length recombinant APOL1 induce ideally cation-selective macroscopic conductances in planar lipid bilayers. The conductances were highly sensitive to pH: their induction required acidic pH (pH 5.3), but their magnitude could be increased 3,000-fold upon alkalinization of the milieu (pK(a) = 7.1). We show that this phenomenon can be attributed to the association of APOL1 with the bilayer at acidic pH, followed by the opening of APOL1-induced cation-selective channels upon pH neutralization. Furthermore, the conductance increase at neutral pH (but not membrane association at acidic pH) was prevented by the interaction of APOL1 with the serum resistance-associated protein, which is produced by T. brucei rhodesiense and prevents trypanosome lysis by APOL1. These data are consistent with a model of lysis that involves endocytic recycling of APOL1 and the formation of cation-selective channels, at neutral pH, in the parasite plasma membrane.

Synthesis, Crystal Structure, and Photoluminescent Properties of 3,3',4,4'-Tetraethyl-5,5'-divinyl-2,2'-bipyrrole Derivatives.

Photoluminescent divinylbipyrroles were synthesized from 3,3',4,4'-tetraetyl-2,2'-bipyrrole-5,5'-dicarboxaldehyde and activated methylene compounds via aldol condensation. For mechanistic clarity, molecular structures of Meldrum's acid- and 1,3-dimethylbarbituric acid-derived divinylbipyrroles were determined by single-crystal X-ray diffraction. Photoluminescent properties of the synthesized divinylbipyrroles in dichloromethane were found to be dependent on the presence of electron withdrawing groups at the vinylic terminal. The divinylbipyrroles derived from malononitrile, Meldrum's acid, and 1,3-dimethylbarbituric acid showed fluorescent peaks at 553, 576, and 602 nm respectively. Computational studies indicated that the alkyl substituents on the bipyrrole 3 and 3' positions increased energy level of the highest occupied molecular orbital (HOMO) compared to the unsubstituted derivatives and provided rationale for the bathochromic shift of the ultraviolet-visible (UV-Vis) spectra compared to the previously reported analogs.

Topography of the TH5 Segment in the Diphtheria Toxin T-Domain Channel.

The translocation domain (T-domain) of diphtheria toxin contains 10 α helices in the aqueous crystal structure. Upon exposure to a planar lipid bilayer under acidic conditions, it inserts to form a channel and transport the attached amino-terminal catalytic domain across the membrane. The TH5, TH8, and TH9 helices form transmembrane segments in the open-channel state, with TH1-TH4 translocated across the membrane. The TH6-TH7 segment also inserts to form a constriction that occupies only a small portion of the total channel length. Here, we have examined the TH5 segment in more detail, using the substituted-cysteine accessibility method. We constructed a series of 23 mutant T-domains with single cysteine residues at positions in and near TH5, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents added to the solutions on either side of the membrane. For 15 of the mutants, the reagent caused a decrease in single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. We also found that reaction caused large changes in ionic selectivity for some mutant channels. We determined whether reaction occurred in the open state or in the brief flicker-closed state of the channel. Finally, we compared the reaction rates from either side of the membrane. Our experiments are consistent with the hypotheses that the TH5 helix has a transmembrane orientation and remains helical in the open-channel state; they also indicate that the middle of the helix is aligned with the constriction in the channel.

Association of Extent of Transverse Sinus Stenosis With Cerebral Glymphatic Clearance in Patients With Idiopathic Intracranial Hypertension.

BACKGROUND AND OBJECTIVES: Idiopathic intracranial hypertension (IIH) is a neurologic disorder characterized by symptoms of elevated intracranial pressure in the absence of a clear cause. There is a developing theory that IIH may, in part, be related to abnormal cerebral glymphatic clearance. In addition, transverse sinus stenosis (TSS) is a common finding in IIH of unclear pathophysiologic significance. Similarly, whether or not TSS is associated with glymphatic outflow in IIH is unknown. The aim of this investigation was to explore the possible association between glymphatic outflow and extent of TSS in patients with IIH. METHODS: The study cohort consisted of patients with IIH and healthy controls who were retrospectively identified from our tertiary care institution located in upstate New York from 2016 to 2023. Patients with IIH were included if they had brain MRIs completed with sufficient sequences for analysis. Brain MRIs were computationally analyzed using diffusion tensor imaging analysis along the perivascular space technique to quantify the glymphatic function in patients with IIH. Glymphatic clearance, the primary outcome, was then correlated with the degree of TSS on MR venography using 2 different scoring systems, the 'Farb score' and 'Carvalho score.' RESULTS: Overall, 81 patients with IIH (70 [86%] female, mean age 29.8 years [SD: 8.2 years], mean BMI 41 [SD: 8.4]) and 10 normal controls were identified with sufficient imaging. Based on the Carvalho TSS score, IIH patients without TSS had significantly lower glymphatic clearance than healthy controls (mean ALPS index: 1.196 [SD: 0.05] vs 1.238 [SD: 0.04], respectively; p = 0.018). Furthermore, IIH patients with TSS had significantly lower glymphatic outflow than healthy controls (1.129 [SD: 0.07] vs 1.238 [SD: 0.04], respectively; p < 0.0001) and IIH patients without TSS (1.129 [SD: 0.07] vs 1.196 [SD: 0.05], respectively; p < 0.0001). In addition, there was a significant association between increasing extent of TSS and declining glymphatic clearance (p < 0.0001, R = 0.62). Finally, IIH patients with severe TSS had significantly lower glymphatic flow than IIH patients with mild stenosis (1.121 [SD: 0.07] vs 1.178 [SD: 0.05], respectively; p < 0.0001). These findings were similarly recapitulated using the Farb TSS scoring system. DISCUSSION: These preliminary findings suggest that the extent of TSS is associated with the degree of glymphatic clearance in IIH, providing novel insights into IIH pathophysiology. Further research is required to clarify the possible causal relationship between TSS and impaired glymphatic clearance in IIH.

Improved Cerebral Glymphatic Flow after Transvenous Embolization of CSF-Venous Fistula.

Spontaneous intracranial hypotension is characterized by symptoms of low intracranial CSF volume due to various mechanisms of CSF leakage. One such mechanism is a CSF-venous fistula, treatable with transvenous embolization resulting in substantial radiographic and clinical improvement. However, the exact mechanisms underlying these improvements, including the potential involvement of the glymphatic system, remain unclear. To noninvasively assess glymphatic clearance in spontaneous intracranial hypotension, we used an advanced MR imaging technique called the DTI along the perivascular spaces in 3 patients with CSF-venous fistula before and after embolization. All 3 patients with spontaneous intracranial hypotension initially had low glymphatic flow, which improved postembolization. Two patients with symptomatic improvement exhibited a more substantial increase in glymphatic flow compared with a patient with minimal improvement. These findings suggest a possible link between cerebral glymphatics in spontaneous intracranial hypotension pathophysiology and symptomatic improvement, warranting larger studies to explore the role of the glymphatic system in spontaneous intracranial hypotension.

Diffusion-Weighted Imaging Reveals Impaired Glymphatic Clearance in Idiopathic Intracranial Hypertension.

BACKGROUND AND PURPOSE: The pathophysiology underlying idiopathic intracranial hypertension (IIH) remains incompletely understood. While one theory postulates impaired cerebral glymphatic clearance in IIH, there is a paucity of methods to quantify glymphatic activity in human brains. The purpose of this study was to use advanced diffusion-weighed imaging to evaluate the glymphatic clearance of IIH patients and how it may relate to clinical severity. MATERIALS AND METHODS: DWI was used to separately evaluate the diffusivity along the cerebral perivascular spaces and lateral association and projection fibers, with the degree of diffusivity used as a surrogate for glymphatic function (diffusion tensor image analysis along the perivascular space. Patients with IIH were compared with normal controls. Glymphatic clearance was correlated with several clinical metrics, including lumbar puncture opening pressure and Frisen papilledema grade (low grade: 0-2; high grade: 3-5). RESULTS: In total, 99 patients with IIH were identified and compared with 6 healthy controls. Overall, patients with IIH had significantly lower glymphatic clearance based on DWI-derived diffusivity compared with controls (P = .005). Additionally, in patients with IIH, there was a significant association between declining glymphatic clearance and increasing Frisen papilledema grade (P = .046) but no correlation between opening pressure and glymphatic clearance (P = .27). Furthermore, healthy controls had significantly higher glymphatic clearance compared with patients with IIH and low-grade papilledema (P = .015) and high-grade papilledema (P = .002). Lastly, patients with IIH and high-grade papilledema had lower glymphatic clearance compared with patients with IIH and low-grade papilledema (P = .005). CONCLUSIONS: Patients with IIH possess impaired glymphatic clearance, which is directly related to the extent of clinical severity. The DWI-derived parameters can be used for clinical diagnosis or to assess response to treatment.

Total brain volume is associated with severity of transverse sinus stenosis in idiopathic intracranial hypertension.

BACKGROUND: Idiopathic intracranial hypertension (IIH) is a complex neurological condition characterized by symptoms of increased intracranial pressure of unclear etiology. While transverse sinus stenosis (TSS) is often present in patients with IIH, how and why it occurs remains unclear. METHODS: IIH patients and a set of age-matched normal controls were identified from our single-center tertiary care institution from 2016 to 2024. Brain MRIs before treatment were computationally segmented and parcellated using FreeSurfer software. Extent of TSS on MR venograms was graded using the Farb scoring system. Relationship between normalized brain volume, normalized brain-to-CSF volume, and TSS was investigated. Multiple linear regression was conducted to investigate the association between continuous variables, accounting for the covariates body mass index, sex, and age. RESULTS: In total, 84 IIH patients (mean age, 29.8 years; 87% female) and 15 normal controls (mean age, 28.1 years) were included. Overall, increasing/worsening TSS was found to be significantly associated with normalized total brain volume (p=0.018, R=0.179) and brain-to-CSF ratio volume (p=0.026, R=0.184). Additionally, there was a significant difference between controls and IIH patients with mild and severe stenosis regarding normalized total brain volume (ANCOVA, p=0.023) and brain-to-CSF ratio volume (ANCOVA, p=0.034). Likewise, IIH patients with severe TSS had a significantly higher brain-to-CSF volume compared with controls (p=0.038) and compared with IIH patients with mild TSS (p=0.038). CONCLUSIONS: These findings suggest that total brain volume is associated with extent of TSS, which may reflect extramural venous compression due to enlarged brain and/or venous hypertension with associated cerebral congestion/swelling.

Crucial role of H322 in folding of the diphtheria toxin T-domain into the open-channel state.

The translocation (T) domain plays a key role in the entry of diphtheria toxin into the cell. Upon endosomal acidification, the T-domain undergoes a series of conformational changes that lead to its membrane insertion and formation of a channel. Recently, we have reported that the triple replacement of C-terminal histidines H322, H323, and H372 with glutamines prevents the formation of open channels in planar lipid bilayers. Here, we report that this effect is primarily due to the mutation of H322. We further examine the relationship between the loss of functionality and membrane folding in a series of mutants with C-terminal histidine substitutions using spectroscopic assays. The membrane insertion pathway for the mutants differs from that of the wild type as revealed by the membrane-induced red shift of tryptophan fluorescence at pH 6.0-6.5. T-Domain mutants with replacements at H323 and H372, but not at H322, regain a wild-type-like spectroscopic signature upon further acidification. Circular dichroism measurements confirm that affected mutants misfold during insertion into vesicles. Conductance measurements reveal that substituting H322 dramatically reduces the numbers of properly folded channels in a planar bilayer, but the properties of the active channels appear to be unaltered. We propose that H322 plays an important role in the formation of open channels and is involved in guiding the proper insertion of the N-terminal region of the T-domain into the membrane.

Cognitive and neuroimaging outcomes in individuals with benign and low-grade brain tumours receiving radiotherapy: a protocol for a prospective cohort study.

INTRODUCTION: Radiation-induced cognitive decline (RICD) occurs in 50%-90% of adult patients 6 months post-treatment. In patients with low-grade and benign tumours with long expected survival, this is of paramount importance. Despite advances in radiation therapy (RT) treatment delivery, better understanding of structures important for RICD is necessary to improve cognitive outcomes. We hypothesise that RT may affect network topology and microstructural integrity on MRI prior to any gross anatomical or apparent cognitive changes. In this longitudinal cohort study, we aim to determine the effects of RT on brain structural and functional integrity and cognition. METHODS AND ANALYSIS: This study will enroll patients with benign and low-grade brain tumours receiving partial brain radiotherapy. Patients will receive either hypofractionated (>2 Gy/fraction) or conventionally fractionated (1.8-2 Gy/fraction) RT. All participants will be followed for 12 months, with MRIs conducted pre-RT and 6-month and 12 month post-RT, along with a battery of neurocognitive tests and questionnaires. The study was initiated in late 2018 and will continue enrolling through 2024 with final follow-ups completing in 2025. The neurocognitive battery assesses visual and verbal memory, attention, executive function, processing speed and emotional cognition. MRI protocols incorporate diffusion tensor imaging and resting state fMRI to assess structural connectivity and functional connectivity, respectively. We will estimate the association between radiation dose, imaging metrics and cognitive outcomes. ETHICS AND DISSEMINATION: This study has been approved by the Research Subjects Review Board at the University of Rochester (STUDY00001512: Cognitive changes in patients receiving partial brain radiation). All results will be published in peer-reviewed journals and at scientific conferences. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT04390906.

Direct percutaneous puncture versus transarterial embolization for head and neck paragangliomas: A systematic review and meta-analysis.

INTRODUCTION: Embolization of head and neck paragangliomas (HNPs) is a well-established treatment strategy and adjunctive therapy. However, the optimal mode of intervention, whether by direct percutaneous puncture (DP) or via transarterial embolization (TAE), remains unclear. METHODS: The aim of this study was to complete a systematic literature review and meta-analysis to compare the safety and efficacy of DP versus TAE for HNP embolization. The Cochrane Library and MEDLINE databases were used to identify studies describing the clinical outcomes of either DP or TAE for HNP embolization. Outcome measures included: complete angiographic devascularization, major complications, and minor complications. Pooled rates were calculated for each variable which were then compared with meta-regression using a random effects model. RESULTS: Thirty-one retrospective studies met inclusion criteria, detailing 394 patients with 411 HNPs. Overall, DP was associated with a higher rate of complete devascularization (91.5%, 95% confidence interval [CI]: 85.6% to 97.4%; I2 = 0%) when compared to TAE technique (40.1%, CI: 27.2% to 58.9%; I2 = 93%). However, there was no difference regarding major complication rates between DP (6%, CI:1.3% to 10.8%; I2 = 0%) and TAE for HNP embolization (3.3%, CI: 1.4% to 5.3%; I2 = 0%) (p = 0.370), nor in minor complications between the techniques (p = 0.211). Subgroup analysis of TAE embolic agents revealed that particle embolics were associated with a significantly lower rate of major complications (2.5%; 0.4% to 4.6%; I2 = 0%) when compared to liquid embolics (10.6%, CI:4% to 17.3%; I2 = 48%; p = 0.022). CONCLUSIONS: A DP approach for HNP embolization results in a higher rate of complete devascularization and with a similar complication profile when compared to TAE. These findings also suggest that particle embolics are associated with fewer major complications compared to liquid embolics when TAE is utilized.

Association of Radiation Dose to the Amygdala-Orbitofrontal Network with Emotion Recognition Task Performance in Patients with Low-Grade and Benign Brain Tumors.

BACKGROUND: Although data are limited, difficulty in social cognition occurs in up to 83% of patients with brain tumors. It is unknown whether cranial radiation therapy (RT) dose to the amygdala-orbitofrontal network can impact social cognition. METHODS: We prospectively enrolled 51 patients with low-grade and benign brain tumors planned for cranial RT. We assessed longitudinal changes on an emotion recognition task (ERT) that measures the ability to recognize emotional states by displaying faces expressing six basic emotions and their association with the RT dose to the amygdala-orbitofrontal network. ERT outcomes included the median time to choose a response (ERTOMDRT) or correct response (ERTOMDCRT) and total correct responses (ERTHH). RESULTS: The RT dose to the amygdala-orbitofrontal network was significantly associated with longer median response times on the ERT. Increases in median response times occurred at lower doses than decreases in total correct responses. The medial orbitofrontal cortex was the most important variable on regression trees predicting change in the ERTOMDCRT. DISCUSSION: This is, to our knowledge, the first study to show that off-target RT dose to the amygdala-orbitofrontal network is associated with performance on a social cognition task, a facet of cognition that has previously not been mechanistically studied after cranial RT.

TonB-dependent transporter FhuA in planar lipid bilayers: partial exit of its plug from the barrel.

TonB-dependent transporters (TBDTs), which transport iron-chelating siderophores and vitamin B(12) across the outer membrane of Gram-negative bacteria, share a conserved architecture of a 22-stranded ß-barrel with an amino-terminal plug domain occluding the barrel. We previously reported that we could induce TBDTs to reversibly open in planar lipid bilayers via the use of urea and that these channels were responsive to physiological concentrations of ligands. Here we report that in the presence of urea, trypsin can cleave the amino-terminal 67 residues of the plug of the TonB-dependent transporter FhuA, as assessed by gel shift and mass spectrometry assays. On the bilayer, trypsin treatment in the presence of urea resulted in the induced conductance no longer being reversed upon removal of urea, suggesting that urea opens intact FhuA channels by pulling the plug at least partly out of the barrel and that removal of the urea then allows reinsertion of the plug into the barrel. When expressed separately, the FhuA plug domain was found to be a mostly unfolded structure that was able to occlude isolated FhuA ß-barrels inserted into the membrane. Thus, although folded in the barrel, the plug need not be folded upon exiting the barrel. The rate of insertion of the ß-barrels into the membrane was tremendously increased in the presence of an osmotic gradient provided by either urea or glycerol. Negative staining electron microscopy showed that FhuA in a detergent solution formed vesicles, thus explaining why an osmotic gradient promoted the insertion of FhuA into membranes.

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes.

Micronutrients such as siderophore-bound iron and vitamin B(12) cross the outer membrane of gram-negative bacteria through a group of 22-stranded beta-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the beta-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ion-conducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellular-facing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.

Longitudinal Effects of Combination Antiretroviral Therapy on Cognition and Neuroimaging Biomarkers in Treatment-Naive People With HIV.

BACKGROUND AND OBJECTIVES: While combination antiretroviral therapy (cART) has dramatically increased the life expectancy of people with HIV (PWH), nearly 50% develop HIV-associated neurocognitive disorders. This may be due to previously uncontrolled HIV viral replication, immune activation maintained by residual viral replication or activation from other sources, or cART-associated neurotoxicity. The aim of this study was to determine the effect of cART on cognition and neuroimaging biomarkers in PWH before and after initiation of cART compared with that in HIV-negative controls (HCs) and HIV elite controllers (ECs) who remain untreated. METHODS: We recruited 3 groups of participants from the University of Rochester, McGovern Medical School, and SUNY Upstate Medical University: (1) ART treatment-naive PWH; (2) age-matched HCs; and (3) ECs. Participants underwent brain MRI and clinical and neuropsychological assessments at baseline, 1 year, and 2 years. PWH were also assessed 12 weeks after initiating cART. Volumetric analysis and fractal dimensionality (FD) were calculated for cortical and subcortical regions. Mixed effect regressions examined the effect of group and imaging variables on cognition. RESULTS: We enrolled 47 PWH, 58 HCs, and 10 ECs. At baseline, PWH had worse cognition and lower cortical volumes than HCs. Cognition improved after initiation of cART and remained stable over time. Greater cortical thickness was associated with better cognition at baseline; greater FD of parietal, temporal, and occipital lobes was associated with better cognition at baseline and longitudinally. At baseline, ECs had worse cognition, lower cortical thickness, and lower FD in all 4 lobes and caudate than PWH and HCs. Greater cortical thickness, hippocampal volumes, and FD of frontal, temporal, and occipital lobes were associated with better cognition longitudinally. DISCUSSION: Initiation of cART in PWH is associated with improvement in brain structure and cognition. However, significant differences persist over time when compared with HCs. Similar trends in ECs suggest that results are due to HIV infection rather than treatment. Stronger associations between cognition and FD suggest this imaging metric may be a more sensitive marker of neuronal injury than cortical thickness and volumetric measures.

Replacement of C-terminal histidines uncouples membrane insertion and translocation in diphtheria toxin T-domain.

The translocation (T) domain plays a key role in the action of diphtheria toxin and is responsible for transferring the N-terminus-attached catalytic domain across the endosomal membrane into the cytosol in response to acidification. The T-domain undergoes a series of pH-triggered conformational changes that take place in solution and on the membrane interface, and ultimately result in transbilayer insertion and N-terminus translocation. Structure-function studies along this pathway have been hindered because the protein population occupies multiple conformations at the same time. Here we report that replacement of the three C-terminal histidine residues, H322, H323, and H372, in triple-R or triple-Q mutants prevents effective translocation of the N-terminus. Introduction of these mutations in the full-length toxin results in decrease of its potency. In the context of isolated T-domain, these mutations cause loss of characteristic conductance in planar bilayers. Surprisingly, these mutations do not affect general folding in solution, protein interaction with the membranes, insertion of the consensus transmembrane helical hairpin TH8-9, or the ability of the T-domain to destabilize vesicles to cause leakage of fluorescent markers. Thus, the C-terminal histidine residues are critical for the transition from the inserted intermediate state to the open-channel state in the insertion/translocation pathway of the T-domain.

The colicin Ia receptor, Cir, is also the translocator for colicin Ia.

Colicin Ia, a channel-forming bactericidal protein, uses the outer membrane protein, Cir, as its primary receptor. To kill Escherichia coli, it must cross this membrane. The crystal structure of Ia receptor-binding domain bound to Cir, a 22-stranded plugged beta-barrel protein, suggests that the plug does not move. Therefore, another pathway is needed for the colicin to cross the outer membrane, but no 'second receptor' has ever been identified for TonB-dependent colicins, such as Ia. We show that if the receptor-binding domain of colicin Ia is replaced by that of colicin E3, this chimera effectively kills cells, provided they have the E3 receptor (BtuB), Cir, and TonB. This is consistent with wild-type Ia using one Cir as its primary receptor (BtuB in the chimera) and a second Cir as the translocation pathway for its N-terminal translocation (T) domain and its channel-forming C-terminal domain. Deletion of colicin Ia's receptor-binding domain results in a protein that kills E. coli, albeit less effectively, provided they have Cir and TonB. We show that purified T domain competes with Ia and protects E. coli from being killed by it. Thus, in addition to binding to colicin Ia's receptor-binding domain, Cir also binds weakly to its translocation domain.

Fixel-Based Analysis and Free Water Corrected DTI Evaluation of HIV-Associated Neurocognitive Disorders.

Background: White matter (WM) damage is a consistent finding in HIV-infected (HIV+) individuals. Previous studies have evaluated WM fiber tract-specific brain regions in HIV-associated neurocognitive disorders (HAND) using diffusion tensor imaging (DTI). However, DTI might lack an accurate biological interpretation, and the technique suffers from several limitations. Fixel-based analysis (FBA) and free water corrected DTI (fwcDTI) have recently emerged as useful techniques to quantify abnormalities in WM. Here, we sought to evaluate FBA and fwcDTI metrics between HIV+ and healthy controls (HIV-) individuals. Using machine learning classifiers, we compared the specificity of both FBA and fwcDTI metrics in their ability to distinguish between individuals with and without cognitive impairment in HIV+ individuals. Methods: Forty-two HIV+ and 52 HIV- participants underwent MRI exam, clinical, and neuropsychological assessments. FBA metrics included fiber density (FD), fiber bundle cross section (FC), and fiber density and cross section (FDC). We also obtained fwcDTI metrics such as fractional anisotropy (FAT) and mean diffusivity (MDT). Tract-based spatial statistics (TBSS) was performed on FAT and MDT. We evaluated the correlations between MRI metrics with cognitive performance and blood markers, such as neurofilament light chain (NfL), and Tau protein. Four different binary classifiers were used to show the specificity of the MRI metrics for classifying cognitive impairment in HIV+ individuals. Results: Whole-brain FBA showed significant reductions (up to 15%) in various fiber bundles, specifically the cerebral peduncle, posterior limb of internal capsule, middle cerebellar peduncle, and superior corona radiata. TBSS of fwcDTI metrics revealed decreased FAT in HIV+ individuals compared to HIV- individuals in areas consistent with those observed in FBA, but these were not significant. Machine learning classifiers were consistently better able to distinguish between cognitively normal patients and those with cognitive impairment when using fixel-based metrics as input features as compared to fwcDTI metrics. Conclusion: Our findings lend support that FBA may serve as a potential in vivo biomarker for evaluating and monitoring axonal degeneration in HIV+ patients at risk for neurocognitive impairment.

Protein translocation through the anthrax toxin transmembrane pore is driven by a proton gradient.

Protective antigen (PA) from anthrax toxin assembles into a homoheptamer on cell surfaces and forms complexes with the enzymatic components: lethal factor (LF) and edema factor (EF). Endocytic vesicles containing these complexes are acidified, causing the heptamer to transform into a transmembrane pore that chaperones the passage of unfolded LF and EF into the cytosol. We show in planar lipid bilayers that a physiologically relevant proton gradient (DeltapH, where the endosome is acidified relative to the cytosol) is a potent driving force for translocation of LF, EF and the LF amino-terminal domain (LFN) through the PA63 pore. DeltapH-driven translocation occurs even under a negligible membrane potential. We found that acidic endosomal conditions known to destabilize LFN correlate with an increased translocation rate. The hydrophobic heptad of lumen-facing Phe427 residues in PA (or phi clamp) drives translocation synergistically under a DeltapH. We propose that a Brownian ratchet mechanism proposed earlier for the phi clamp is cooperatively linked to a protonation-state, DeltapH-driven ratchet acting trans to the phi-clamp site. In a sense, the channel functions as a proton/protein symporter.

Sizing the protein translocation pathway of colicin Ia channels.

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include /=90 mV, even a 26-A stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is >/=26 A in diameter, or can stretch to this value. This is large enough for an alpha-helical hairpin to fit through.