Alzheimer’s disease (AD) and other tauopathies are characterized by the aggregation of tau into soluble and insoluble forms (including tangles and neuropil threads). In humans, a fraction of both phosphorylated and non-phosphorylated N-terminal to mid-domain tau species, are secreted into cerebrospinal fluid (CSF). Some of these CSF tau species can be measured as diagnostic and prognostic biomarkers, starting from early stages of disease. While in animal models of AD pathology, soluble tau aggregates have been shown to disrupt neuronal function, it is unclear whether the tau species present in CSF will modulate neural activity. Here, we have developed and applied a novel approach to examine the electrophysiological effects of CSF from patients with a tau-positive biomarker profile. The method involves incubation of acutely-isolated wild-type mouse hippocampal brain slices with small volumes of diluted human CSF, followed by a suite of electrophysiological recording methods to evaluate their effects on neuronal function, from single cells through to the network level. Comparison of the toxicity profiles of the same CSF samples, with and without immuno-depletion for tau, has enabled a pioneering demonstration that CSF-tau potently modulates neuronal function. We demonstrate that CSF-tau mediates an increase in neuronal excitability in single cells. We then observed, at the network level, increased input–output responses and enhanced paired-pulse facilitation as well as an increase in long-term potentiation. Finally, we show that CSF-tau modifies the generation and maintenance of hippocampal theta oscillations, which have important roles in learning and memory and are known to be altered in AD patients. Together, we describe a novel method for screening human CSF-tau to understand functional effects on neuron and network activity, which could have far-reaching benefits in understanding tau pathology, thus allowing for the development of better targeted treatments for tauopathies in the future.The online version contains supplementary material available at 10.1186/s40478-023-01562-5. Tau plays a central role in the neuropathology of Alzheimer's disease (AD) and other tauopathies , 113. WhIn AD, N-terminal to mid-domain tau fragments, both phosphorylated and non-phosphorylated variants, are released into the cerebrospinal fluid (CSF) early in disease progression, which has enabled the development of biomarkers for disease prognosis, diagnosis, and staging , 111. WhWe have previously developed a technique using whole-cell patch-clamp recording to introduce structurally-defined soluble full-length recombinant tau aggregates into single neurons –62. UsinActive cortical networks support several rhythms (such as gamma and theta oscillations) that can be measured clinically including in individuals suspected to be affected by AD. Theta oscillations (4–7 Hz) represent the largest extracellular signal recorded in the mammalian brain  and are In this study, we have characterised the functional neurotoxic changes induced by tau-positive CSF samples, on neuronal function. We have used a suite of detailed electrophysiological measures; from single cells to network activity. We demonstrate that CSF-tau mediates an increase in neuronal excitability, alters synaptic transmission and plasticity and modifies the generation and maintenance of hippocampal theta oscillations. However, the same CSF samples, with tau removed by immuno-depletion, failed to produce these effects, indicating that tau is central to these neural changes.All CSF samples were obtained from patients by lumbar puncture from the L3/L4 or the L4/L5 inter-space in the morning, according to standardized procedure. Samples were collected in a propylene tube and centrifuged for 10 min at 1800 × g at + 4 °C. The supernatant was stored at − 80 °C prior to use. The CSF samples used in this study were the remainder following clinical routine analyses, from multiple de-identified patients, pooled together in a procedure approved by the Ethics Committee at the University of Gothenburg (#EPN 140811). The collection and use of CSF samples were in accordance with the Swedish law of biobank in healthcare (2002:297).The biomarker profile for the pool, including Aβ42, Aβ40, total tau (t-tau) and phosphorylated tau at threonine 181 (p-tau181), was measured with the fully automated Lumipulse (Fujirebio) platform following published protocols . The CSFCSF collection and processing were performed at the University of Gothenburg, Sweden, with local ethical approval, and the samples sent to the University of Warwick, UK, where all experiments were done as approved by the local Human Tissue Authority and Biomedical & Scientific Research Ethics Committees.2+, low Ca2+ artificial CSF (aCSF), composed of the following: 127 mM NaCl, 1.9 mM KCl, 8 mM MgCl2, 0.5 mM CaCl2, 1.2 mM KH2PO4, 26 mM NaHCO3, and 10 mM D-glucose . Slices were stored at 34 °C in standard aCSF (1 mM Mg2+ and 2 mM Ca2+) for between 1 and 8 h.All animal care and experimental procedures were reviewed and approved by the institutional animal welfare and ethical review body (AWERB) at the University of Warwick. Animals were kept in standard housing with littermates, provided with food and water ad libitum and maintained on a 12:12 (light–dark) cycle. C57BL/6 mice  were killed by cervical dislocation and decapitated in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986). The brain was rapidly removed, and acute parasagittal or horizontal brain slices (350–400 μm) were cut with a Microm HM 650 V microslicer in cold (2–4 °C) high Mg2, 5% CO2 using microloaders (Eppendorf). Slices were placed into the incubation chambers one at a time (minimum volume 1.5 ml to cover raised grid). This pre-incubation protocol is an efficient method of exposing acute mouse brain slices to small volumes of diluted human CSF. The CSF sample volumes in this study and proposed for future studies  will not allow for perfusion of the diluted CSF through the recording rig (requiring at least 30–40 ml of solution). Therefore, following incubation individual slices were placed on the recording rig and perfused with regular aCSF throughout the recording period, so the CSF-tau was only present for the 1-h incubation. In further control experiments, slices were incubated in either CSF-mock-depleted (immuno-depletion control using a combination of anti-mouse IgG and neurogranin antibodies) or CSF from a control patient  to further validate our findings  or in varying dilutions  of CSF-tau or CSF-tau-depleted (immunodepleted for tau) for 1 h, in bespoke incubation chambers at room temperature. The incubation chambers consisted of small, raised grids  placed in the wells of a twenty-four well plate  which were bubbled with 95% OA slice was transferred to the recording chamber, submerged, and perfused (2–3 ml/min) with aCSF at 30 °C. Slices were visualised using IR-DIC optics with an Olympus BX151W microscope (Scientifica) and a CCD camera (Hitachi). Whole-cell current-clamp recordings were made from pyramidal cells in area CA1 of the hippocampus using patch pipettes (5–10 MΩ) manufactured from thick-walled glass (Harvard Apparatus). Pyramidal cells were identified by their position in the slice, morphology (from fluorescence imaging) and characteristics of the standard step current − voltage relationship. Voltage recordings were made using an Axon Multiclamp 700B amplifier (Molecular Devices) and digitised at 20 kHz. Data acquisition and analysis were performed using pClamp 10 (Molecular Devices). Recordings from neurons that had a resting membrane potential of between − 55 and − 75 mV at whole-cell breakthrough were accepted for analysis. The bridge balance was monitored throughout the experiments and any recordings where it changed by > 20% were discarded.To extract the electrophysiological properties of recorded neurons, both step, ramp and more naturalistic, fluctuating currents were injected.The standard current–voltage relationship was constructed by injecting standard (step) currents from − 200 pA incrementing by either 50 or 100 pA (1 s duration) until a regular firing pattern was induced. A plot of step current against voltage response around the resting potential was used to measure the input resistance (from the gradient of the fitted line).To evaluate the rheobase ) a current ramp was injected into neurons. From the baseline, a 100 ms duration ramp to − 100 pA was followed by a 900 ms duration ramp at 0.33 pA/ms up to 200 pA, then a step back down to baseline (zero current).τfast = 3 ms and τslow = 10 ms. This current waveform, which mimics the stochastic actions of AMPA and GABA-receptor channel activation, is injected into cells and the resulting voltage recorded . The voltage trace was used to measure the frequency of action potential firing and to construct a dynamic-I–V curve. The firing rate was measured from voltage traces evoked by injecting a current wave form of the same gain for all recordings (to give a firing rate of ∼2–3 Hz). Action potentials were detected by a manually set threshold and the interval between action potentials measured. Dynamic I–V curves were constructed and used to extract a number of parameters including the capacitance, time constant, input resistance, resting membrane potential, spike threshold and spike onset , filtered at 3 kHz and digitised at 20 kHz . Data acquisition was performed using pClamp 10 (Molecular Devices). Analysis of mEPSCs was performed using MiniAnalysis software (SynaptoSoft). Events were manually analysed and were accepted if they had amplitude > 6 pA (events below this amplitude were difficult to distinguish from baseline noise) and had a faster rise than decay. Statistical significance was measured using a one-way ANOVA.AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) were recorded as previously described . To isolA 400 μM parasagittal slice was transferred to the submerged recording chamber and perfused with aCSF at 4–6 ml/min (32 °C). The slice was placed on a grid allowing perfusion above and below the tissue and all tubing (Tygon) was gas tight (to prevent loss of oxygen). To record field excitatory postsynaptic potentials (fEPSPs), an aCSF-filled microelectrode was placed on the surface of stratum radiatum in CA1. A bipolar concentric stimulating electrode (FHC) controlled by an isolated pulse stimulator model 2100  was used to evoke fEPSPs at the Schaffer collateral–commissural pathway. Field EPSPs were evoked every 30 s (0.03 Hz). Stimulus input/output curves for fEPSPs were generated using stimulus strength of 1–5 V for all slices (stimulus duration 200 μs). For the synaptic plasticity experiments, the stimulus strength was set to produce a fEPSP slope ~ 40% of the maximum response and a 20-min baseline was recorded before plasticity induction. Paired-pulse facilitation was measured over an interval range of 20 to 500 ms. Long-term potentiation (LTP) was induced by high frequency stimulation  and then fEPSPs were recorded for 60 min following LTP induction. Signals were filtered at 3 kHz and digitised on-line (10 kHz) with a Micro CED (Mark 2) interface controlled by Spike software . The fEPSP slope was measured from a 1 ms linear region following the fibre volley.A 400 µM horizontal slice was transferred to an interface recording chamber (Digitimer BSC3) and placed on a mesh support at the interface of an oxygen-rich atmosphere and underlying aCSF. The temperature of the aCSF was set at 31 °C and the flow rate was 1.5 ml/min. An aCSF-filled glass microelectrode was placed in CA3 to record activity. Baseline activity was recorded for 20 min before the bath application of the acetylcholine receptor agonist carbachol, selected to induce oscillations given the evidenced role of acetylcholine receptors in regulating hippocampal-dependent oscillations , 123. Ca2) were compared using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test. The peak oscillatory power of each trace (n = 8 per group) was also compared with the corresponding baseline (n = 8 per group) within each treatment group and mean differences assessed using Wilcoxon tests. Finally, the mean time period  from application of carbachol to the onset of oscillatory activity was compared between treatment groups using a Kruskal–Wallis test followed by a Dunn’s multiple comparisons test.Carbachol-elicited oscillations were characterised using power spectral density (PSD) analysis in Spike 2. PSD profiles of the field potential recordings filtered for the theta band (4–7 Hz) were generated by Fourier transform analysis . The profiles were calculated from a 100–300 s section of the field potential trace displaying peak oscillatory activity, with the baseline power subtracted offline , carbachol , Tetrodotoxin  and trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline  were made up as stock solutions (1–50 mM) in distilled water or dimethyl sulfoxide (DMSO) and then diluted in aCSF on the day of use. The concentration of DMSO did not exceed 0.1% in the final solutions.Statistical analysis was performed using GraphPad Prism. Due to small sample sizes (n < 15), statistical analysis was performed using non-parametric methods; Kruskal–Wallis analysis of variance (ANOVA), Mann–Whitney and Wilcoxon signed-rank tests as required. All data are represented as mean and standard error of the mean with individual experiments represented by single data points. For all experiments, significance was set at p ≤ 0.05. Data points for each experimental condition were derived from a minimum of four individual animals.To investigate the effects of CSF-tau on neuronal properties, in this proof-of-principle study, we used pooled CSF from multiple de-identified patients. The biomarker profile for the pool was measured with the Lumipulse platform. For the pooled samples used in this study, CSF Aβ1-42 level 469 pg/ml) was abnormal as it was below the clinically validated abnormality threshold of 526 pg/ml . Aβ1-42/ pg/ml waTo allow determination of the specific effects of tau on electrophysiological properties, an aliquot of the pooled CSF-tau sample was immuno-depleted for tau by the simultaneous application of the monoclonal antibodies Tau12, HT7 and TauAB  that together cover a wide span of the tau-441 protein sequence. To confirm that tau removal protocols were effective, the immuno-depleted samples were reanalysed for t-tau, p-tau181 and ptau-231 using Simoa assays. P-tau181, p-tau231 and total-tau signals decreased by 86.9% and 92.4% and 83.8%, respectively. To further validate the depletion, p-tau181 was measured using a fully automated Lumipulse platform and the signal decreased by 80.5%  allowed us to perform a wide range of experiments using the same sample to fully characterise its neurotoxic effects. We diluted the CSF-tau sample with aCSF to reduce the volume of sample that was required for each experiment. As our eventual end goal will be to use individual patient samples  to enable correlation with clinical data, we first sought to find the minimum volume (maximum dilution in aCSF) of CSF-tau, for which we could detect a change in neuronal function. We tested 1:100, 1:30 and 1:15 dilutions of the CSF-tau in aCSF (n = 8 per group). We used neuronal excitability, measured using whole-cell patch clamp recording from single CA1 pyramidal cells in the hippocampus, as the functional readout. We have previously found this to be a reliable and robust change induced by recombinant tau , 61. We To fully evaluate the effects of CSF-tau at a single neuron level, slices were either incubated with CSF-tau (n = 11), CSF-tau-depleted (n = 10) or control aCSF  for 1 h before recordings were made. Initially, standard current step protocols were used to measure passive neuronal parameters . Incubation with CSF-tau led to significant depolarisation of the resting membrane potential . The mean IR in control slices was 143.6 ± 5.2 MΩ, compared to the mean IR in slices incubated in CSF-tau of 167 ± 7.2 MΩ; Fig. Given that CSF-tau depolarises the resting membrane potential and increases the IR, we predicted that it should also increase the action potential firing rate. To test this, we used a 40 s fluctuating naturalistic current injection (to mimic synaptic activation) and measured the firing rate. Incubation with CSF-tau significantly increased firing rate (FR). The mean FR in control was 2.3 ± 0.5 Hz compared to the mean FR in neurons exposed to CSF-tau of 4.2 ± 0.6 Hz; Fig. ; H (2) = 9.3, p < 0.0093), with Dunn’s tests revealing a significant decrease in rheobase following CSF-tau treatment relative to aCSF control slices (p = 0.0080), whilst there was no significant difference between aCSF control and CSF-tau-depleted groups (p = 0.8856).We have previously shown that tau aggregates decrease the rheobase , therefTo determine if other neuronal parameters were significantly altered by CSF-tau, we used the dynamic IV method , 11 to mThus, CSF-tau replicates a number of the pathological effects which have been reported previously in models of tauopathy. These effects were absent when tau was removed by immunodepletion, indicating that tau is critical to these disease-associated changes.Next, we investigated whether the changes to neuronal excitability in single pyramidal cells were accompanied by changes in synaptic transmission and plasticity in the CA1 region of the hippocampus.Incubation in CSF-tau led to increased basal synaptic transmission, as measured by stimulus input/output curves Fig. a, b. ForWe then examined whether there were changes to paired pulse facilitation (PPF). If the increase in fEPSP amplitude is a consequence of an increase in release probability, then it would be predicted that the degree of PPF would be reduced. However, the opposite was observed, and the degree of facilitation was significantly increased at 100 ms and 200 ms intervals Fig. c, d. ForIn the same recordings, we then examined whether CSF-tau altered long-term synaptic plasticity by recording long term potentiation (LTP) at the CA1 Schaffer collateral synapses. We measured the potentiation 60 min after HFS , 137 andA receptors), L689,560 (to block NMDA receptors) and TTX (to block voltage-gated Na channels).Since the increase in fEPSP amplitude did not appear to result from an increase in release probability, we hypothesised that it could result from an increase in synaptic AMPA receptor density. To evaluate whether there are changes in postsynaptic AMPA receptor currents, we used whole-cell voltage clamp recordings from CA1 pyramidal neurons to record AMPA receptor-mediated miniature EPSCs (mEPSCs) in the presence of PTX  in the slices incubated with CSF-tau compared to control slices Fig. a. The diWe also found that the amplitude of mEPSCs was significantly enhanced in CSF-tau incubated slices Fig. d. Using Finally, to establish whether there were any changes to the kinetics of the mEPSCs, we then measured the rise and decay time for the average waveform from each recording. The rise time was measured (10–90%) and the decay time was obtained by fitting with a single exponential. There was no change to rise or decay kinetics between the three conditions.We have found that CSF-tau markedly changes neuronal properties, synaptic transmission, and synaptic plasticity. We have next investigated how these changes can lead to alterations in emergent network activity. Hippocampal hyperexcitability has been identified in AD patients prior to the onset of cognitive symptoms and is associated with increased oscillatory power in theta bands relative to control or MCI groups , 114. Th; H (2) = 10.09, p = 0.0065), with CSF-tau slices showing a significantly quicker onset of oscillations relative to control (p = 0.005) following carbachol application.Bath application of the acetylcholine receptor agonist carbachol 50 µM) induced robust, reliable theta oscillations, as demonstrated by Wilcoxon tests comparing peak oscillatory amplitude and baseline activity for all three treatment groups . We compared this against control aCSF, and the data collected with the CSF-tau (full sample) and CSF-tau-depleted (depleted for tau), see Additional file CSF biomarkers for tau and phosphorylated-tau have proved to be reliable predictors/indicators of AD , 74. TauThe pathological effects of soluble tau aggregates have been studied extensively in animal models , 108. ThTo do this, we incubated acute brain slices from male and female wildtype C57/BL6 3–4-week-old mice with biomarker-quantified tau-positive CSF pooled from multiple patients. We decided to use this age of mouse to ensure that there would be no deficits in the hippocampal neural circuitry (as opposed to using aged mice) where variability between recordings is much higher and thus our baseline parameters would be consistent. Therefore, any changes induced by the CSF-tau would be easy to detect. As the goal for future studies is to scale down the volume of sample from pooled samples (5 ml in this study) to individual patient samples (~ 300 μl), to enable correlation with clinical data, we first determined the minimum volume of CSF-tau (diluted in aCSF) for which we could detect a change to neuronal function. We then performed a suite of electrophysiological tests to evaluate the effect on neuronal and network function. We demonstrate that CSF-tau markedly increases neuronal excitability, alters synaptic transmission and plasticity, and modifies the generation and maintenance of hippocampal theta oscillations.In our experiments, the human CSF was diluted 1:15 in aCSF for slice incubation, which is equivalent to 66 µl of CSF and 934 µl of aCSF , with no human CSF present during recording. Thus, it is extremely unlikely any small changes in the ionic composition of the aCSF plus human aCSF or the presence of neurotransmitters (see ) would hA wide range of tau fragments can be found in CSF. These are mostly N-terminal to mid-domain tau fragments and provide good biomarkers for disease prognosis, diagnosis, and staging , 111. FoWe used both standard IV (with step current injections) and the DIVs  to extract neuronal parameters. In slices incubated with CSF-tau, both approaches for parameterisation measured a marked decrease in whole-cell conductance  and a depolarisation of the resting membrane potential leading to an increase in firing rate . Obtaining comparable results using two independent methods for the extraction of neuronal parameters strengthens the robustness of this observation.Depolarisation of the resting membrane potential and neuronal hyperexcitability are consistent with previously reported data in numerous tauopathy models , 132, foA or GABAB receptors has no effect on PPF at CA1 synapses [2+ levels and that elevated levels of Ca2+ (following the first stimulus) persist for longer durations in neurons exposed to CSF-tau. Thus, facilitation would remain elevated at longer inter-pulse intervals, without effecting the PPF at short intervals. This seems a plausible hypothesis as tau has been linked to changes in intracellular Ca2+ [Soluble tau aggregates have been shown to alter synaptic plasticity both in in vitro and in vivo models of tauopathy , 107. Thsynapses . One coular Ca2+ , 120.As well as observing enhanced PPF, we also observed an increase in LTP magnitude in slices that had been incubated in CSF-tau. Since the baseline fEPSP slope was set to 40–50% maximum, to ensure an equal range for potentiation across the experimental groups, the slope of baseline fEPSPs will have been larger in the CSF-tau group. This could potentially lead to enhanced LTP, since there will be greater synaptic activation, we will look to test this in future studies.We then recorded AMPA receptor-mediated mEPSCs. In slices incubated with CSF-tau, we observed significant increases in both the amplitude and frequency of mEPSCs. Depletion of tau markedly reduced, but did not abolish, the effects of CSF on mEPSC frequency. Thus, either very low levels of tau can produce this effect, or some other component in the CSF is responsible. This will be explored further in future studies. An increase in mEPSC frequency would suggest an increase in release probability, consistent with the increased input–output responses, which could result from an increase in the number of active zones or the number of docked vesicles , 135. ThFunctional theta oscillations are dependent upon synchronised activity within the hippocampus, which in turn is mediated by pyramidal cells and interneurons . ParvalbIn this study, we have demonstrated that incubation of brain slices with CSF-tau markedly enhanced theta oscillations and increased excitatory pyramidal neuron excitability, and that both of these effects were abolished upon immunodepleting for tau. It is currently unclear whether the increase in excitability of pyramidal cells and changes in glutamatergic transmission are sufficient to produce the observed increases in theta power or whether changes in GABAergic inhibition also occurred. Our findings align with existing data in rodent models and AD patients; the deposition of tau in pyramidal cells and PV interneurons from 1 month of age in 3xTg mice was associated with elevated theta oscillations but no behavioural impairments . The impThis leads to the question of what incubation with CSF-tau in this study might be doing to hippocampal neurons to mediate the elevation in the power of theta oscillations. A combination of experimental and simulated data has indicated that the early-stage AD hyperactivity underpinning oscillatory slowing could be due to pyramidal neuron hyperexcitability , 138 andIt has also been demonstrated that changes to theta oscillatory power—similar to what we observed herein—can be used to distinguish between prodromal AD and non-AD cases with cognitive decline . The facTaking all of these changes together, it would be helpful to know whether the changes in membrane potential (4 mV depolarisation), increase in input resistance (~ 1/3) and reduction in rheobase are sufficient to account for the increases in the other parameters measured . This question is difficult to answer without several additional experiments. In this proof of principle study, we only recorded from CA1 pyramidal cells. Thus, we do not know if similar effects on excitability also occur with CA3 pyramidal cells and with inhibitory interneurons. With this additional information it would be possible to construct computational models and for example determine whether these changes could account for the increase in theta oscillations and this will be explored further in future studies.In this study, we have demonstrated that incubation of acutely-isolated wild-type mouse hippocampal brain slices with small volumes of diluted human CSF-tau allowed us to evaluate effects on neuronal function from single cells through to network level effects. Comparison of the toxicity profiles of the same CSF samples, with and without immuno-depletion for tau, enabled a pioneering demonstration that CSF-tau potently modulates neuronal function. We demonstrate that CSF-tau mediates an increase in neuronal excitability in single cells. We observed, at the network level, increased input–output responses and enhanced paired-pulse facilitation as well as an increase in long-term potentiation. Finally, we show that CSF-tau modifies the generation and maintenance of hippocampal theta oscillations, which have important roles in learning and memory and are known to be altered in AD patients. Together, we describe a novel method for screening human CSF to understand functional effects on neuron and network activity, which could have far-reaching benefits in understanding pathological mechanisms of tauopathies, thus allowing the development of better targeted treatments.Additional file 1. Figure 1. Dose response for the effect of CSF-tau on neuronal function. Figure 2. Protocol for analysis of hippocampal CA3 theta oscillations. Figure 3. Validation of tau effects using CSF-mock-depleted.


DNA damage involves in the carcinogenesis of some cancer and may act as a target for therapeutic intervention of cancers. However, it is unclear whether aflatoxin B1 (AFB1)-DNA adducts (ADAs), an important kind of DNA damage caused by AFB1, affect the efficiency of post-operative adjuvant transarterial chemoembolization (po-TACE) treatment improving hepatocellular carcinoma (HCC) survival.A hospital-based retrospective study, including 318 patients with Barcelona Clinic Liver Cancer (BCLC)-C stage HCC from high AFB1 exposure areas, to investigate the potential effects of ADAs in the tissues with HCC on po-TACE treatment. The amount of ADAs in the cancerous tissues was tested by competitive enzyme-linked immunosorbent assay (c-ELISA).P > 0.05).Among these patients with HCC, the average amount of ADAs was 3.00 µmol/mol ± 1.51 µmol/mol DNA in their tissues with cancer. For these patients, increasing amount of ADAs was significantly associated with poorer overall survival (OS) and tumor reoccurrence-free survival (RFS), with corresponding death risk (DR) of 3.69 (2.78–4.91) and tumor recurrence risk (TRR) of 2.95 (2.24–3.88). The po-TACE therapy can efficiently improve their prognosis . Interestingly, this improving role was more noticeable among these patients with high ADAs , but not among those with low ADAs (These results suggest that increasing ADAs in the cancerous tissues may be beneficial for po-TACE in ameliorating the survival of patients with HCC. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high incidence and death rate in the worldwide. Because HCC often displays its’ high early-stage blood metastasis and recurrence risk, patients with HCC usually feature a poor prognosis. With advancing in the scientific technology, some new therapeutic methods, including the transarterial chemoembolization (TACE), radiofrequency ablation, microwave ablation, percutaneous ethanol ablation, targeting therapy and radioembolization, have been developed and utilized in some HCC cases. Among these therapeutic methods, TACE treatment has been reported widely to be able to effectively improve the survival status of patients with HCC, especially these with advanced-stage tumor. In China, post-operative adjuvant TACE (po-TACE) treatment has been recommended as an important post-operative adjuvant treatment for HCC patients with high risk of tumor intrahepatic metastasis. However, increasing evidence has shown that po-TACE treatment displays different therapeutic effects on patients with different genetic profiles or biomarkers. Thus, it is vital to discern specific biomarkers or genetic profiles for improving the therapeutic effects of po-TACE treatment on HCC prognosis.Aflatoxin B1 (AFB1) is an important I-type chemical carcinogen, which can induce HCC . On the However, it is unclear whether ADAs in HCC modify the effects of po-TACE treatment improving HCC survival. A retrospective study was conducted to investigate the association between the amount of ADAs in the cancerous tissues with HCC and the therapeutic effects of po-TACE treatment on HCC.This study was a hospital-based retrospective study from Guangxi area (a high AFB1 exposure area). The inclusion criteria on cases include: (1) HCCs diagnosed by histopathological method; (2) tumor being in the Barcelona Clinic Liver Cancer (BCLC)-C stage; (3) cases having Child-Pugh A-stage liver function; (4) cases receiving partial liver resect or partial liver resect plus po-TACE as initial treatment according to Chinese Manage Criteria of Hepatocarcinoma (CMCH); (5) cases approving of this study and without dropping out; and (6) cases with available clinicopathological data, survival data, and cancerous tissue samples. The exclusion criteria on cases are the following: (1) cases with the history of radiotherapy or chemotherapy before receiving surgical resect therapy; (2) pregnant or breastfeeding women cases; and (3) cases with other tumors except HCC.n = 160).Based on the above-mentioned inclusion and exclusion criteria on cases, a total of 318 HCC patients (including 116 cases previously studied ), were rAfter written consent was obtained, all baseline clinicopathological features of all cases, including gender, age, ethnicity, smoking and drinking status, hepatitis B virus (HBV) and hepatitis C virus (HCV) infective status, liver cirrhosis, tumor grade and stage, and treatment information, were obtained as described previously . At the For survival analyses, all patients with HCC underwent serial monitoring as described previously , 15. In 2, BOLONG, Nanjing Pharmaceutical Factory Co., Ltd, China), doxorubicin  and lipiodol, started 4 weeks after the removal therapy of tumor.In this study, po-TACE was regarded as an adjuvant treatment procedure of the initial therapeutic procedure for eligible patients with HCC as described previously . In brieThe amount of ADAs in the tissues with HCC was calculated by competitive enzyme-linked immunosorbent assay (c-ELISA) as described previously . BrieflyMVD in the tumor was elucidated using immunohistochemistry staining of CD34  as described previously . To analχ2) or t test, whereas the link between ADAs and MVD in the cancerous tissues were analyzed by Spearman’s correlation method. The effects of every variable on HCC survival were elucidated using Kaplan-Meier survival model (with Log-rank test) and Cox regression survival model (including univariable and multivariable models). Median survival time, death risk (DR) and tumor recurrence risk (TRR) were used for evaluating the prognostic potential of variables. In this study, a value of P < 0.05 was considered statistically significant.All statistical analyses were finished by the Statistical Package for Social Science (SPSS) version 18 . The difference between groups was tested by Pearson chi-squared test (P > 0.05).A total of 318 patients with BCLC-C stage HCC were included in the final analyses. The Results from Kaplan-Meier survival models displayed that these HCC patients receiving po-TACE treatment featured a better OS compared to those not doing po-TACE treatment  and RFS vs. 2.92 µmol/mol ± 1.66 µmol/mol DNA) ] and TRR [3.08 (2.36–4.02)] of patients with HCC (The amount of ADAs was elucidated by enzyme-linked immunosorbent assay (ELISA) assay and a similar amount of ADAs was tested in cancerous tissue samples from control and po-TACE group (3.09 µmol/mol ± 1.34 µmol/mol mol DNA) . To analmol DNA)  and RFS mol DNA) . MST andwith HCC . Resultswith HCC .P > 0.05)  , Table 4r = 0.182, P < 0.01).Because of MVD in the tumor tissues positively correlating with therapeutic effects of po-TACE on HCC , this stTo the best of authors’ knowledge, there is not a shred of evidence whether the DNA damage by AFB1 can act as a therapeutic target for HCC. On the basis of a retrospective study, this study investigated the potential association between ADA, an important DNA damage type induced by AFB, and the therapeutic efficiency of po-TACE treatment in improving HCC survival. It was found that increasing the amount of ADAs in the cancerous tissues with HCC had a significant correlation with the therapeutic efficiency of po-TACE on HCC. This study represents the first report indicating the possibility of ADA as a therapeutic target for po-TACE.in vivo and in vitro experiments and clinical trials has shown that TACE treatment can effectively improve the outcomes of patients with HCC [vs. 23.00 months) and MRT (8.00 months vs. 20.00 months) and a smaller DR  and TRR .TACE treatment is an important therapeutic procedure for HCC . Among kwith HCC , 24. Todwith HCC . Additiowith HCC , 26. TheIn the past decade, increasing evidence from clinical practice has shown that the therapeutic efficiency of po-TACE treatment can be modified by individuals’ genetic profiles and some potential biomarkers are identified for predicting the therapeutic efficiency of this treatment. This study investigated the predictive values of a DNA damage biomarker ADA for the effects of po-TACE treatment improving HCC survival through a hospital-based retrospective study and found ADAs in tumor samples with HCC can significantly modify the sensibility of HCC cases on po-TACE treatment. These patients with HADA will obtain a better survival under the conditions of po-TACE therapy than those with LADA. This may be because of the several reasons. First, increasing amount of ADAs in tumor tissues usually indicates the low capacity of detoxicating enzymes , 27, 28.In conclusion, this study describes ADAs in tissues with HCC and its link with the therapeutic value of po-TACE treatment and displays that ADAs can act as a valuable biomarker for forecasting the therapeutic significance of po-TACE treatment. These findings indicate that analyzing the amount of ADAs in the cancerous tissues with HCC may help us form a management strategy to improve the survival of patients with HCC. However, the present study was based on a relatively small sample, which may limit the potential of ADAs predicting the therapeutic efficiency of po-TACE on HCC. Although the present study explored the relationship between ADAs and tumor MVD, the corresponding mechanism analyses were not finished. Thus, forthcoming studies in combination with a larger size sample and mechanism analyses will be required to fully evaluate the utility of ADAs in selecting patients for po-TACE therapy.


Dementia is a prevalent non-motor manifestation among individuals with advanced Parkinson’s disease (PD). Glial fibrillary acidic protein (GFAP) is an inflammatory marker derived from astrocytes. Research has demonstrated the potential of plasma GFAP to forecast the progression to dementia in PD patients with mild cognitive impairment (PD–MCI). However, the predictive role of cerebrospinal fluid (CSF) GFAP on future cognitive transformation and alterations in Alzheimer’s disease (AD)-associated CSF biomarkers in newly diagnosed PD patients has not been investigated.210 de novo PD patients from the Parkinson’s Progression Markers Initiative were recruited. Cognitive progression in PD participants was evaluated using Cox regression. Cross-sectional and longitudinal associations between baseline CSF GFAP and cognitive function and AD-related CSF biomarkers were evaluated using multiple linear regression and generalized linear mixed model.p < 0.001). There was no statistically significant difference in baseline CSF GFAP levels between PD–NC and PD–MCI groups. Higher baseline CSF GFAP predicted greater global cognitive decline over time in early PD patients . Furthermore, Cox regression showed that high baseline CSF GFAP levels were associated with a high risk of developing dementia over an 8-year period in the PD–NC group . In addition, the baseline CSF GFAP was positively correlated with the longitudinal changes of not only CSF α-synuclein , but also CSF biomarkers associated with AD, namely, amyloid-β 42 , total tau  and phosphorylated tau .At baseline, the mean age of PD participants was 60.85 ± 9.78 years, including 142 patients with normal cognition (PD–NC) and 68 PD–MCI patients. The average follow-up time was 6.42 ± 1.69 years. A positive correlation was observed between baseline CSF GFAP and age (β = 0.918, CSF GFAP may be a valuable prognostic tool that can predict the severity and progression of cognitive deterioration, accompanied with longitudinal changes in AD-associated pathological markers in early PD.The online version contains supplementary material available at 10.1186/s12974-023-02843-5. Parkinson’s disease (PD) is a chronic neurodegenerative disorder with diverse etiologies and clinical presentations . Among tThe pathophysiological mechanisms that contribute to cognitive disorder in PD are intricate . Kouli eGlial fibrillary acidic protein (GFAP), a type-III intermediate filament that is predominantly expressed by astrocytes, serves as an indicator of reactive astrogliosis. Its expression is substantially elevated in neurodegenerative illness, including Alzheimer’s disease (AD) and PD –14. PereSeveral post-mortem investigations have evidenced that the aggregation of Lewy bodies composed of α-synuclein (α-syn) within the limbic system and neocortex represents a principal contributor to cognitive dysfunction in PD , 19. SimCSF is the preferred biomarker source for research compared to blood owing to its proximity to the brain structure and its ability to accurately reflect the pathological processes taking place in the brain . TherefoResearch data were downloaded from the Parkinson's Progression Markers Initiative (PPMI) repository . The PPMPD subjects were recruited based on the inclusion criteria: individuals must be 30 years or older at diagnosis; have bradykinesia combined with resting tremor, rigidity, or only asymmetric resting tremor or bradykinesia; be untreated for PD, particularly without medications that might interfere with dopamine transporter imaging or CSF collection; and be free of dementia at baseline (MoCA ≥ 22). To prevent misdiagnosis, the researchers reviewed the diagnosis longitudinally. Patients diagnosed with conditions other than primary PD were excluded from the follow-up. In addition, our investigation mandated that all subjects possess baseline CSF GFAP data. All enrolled participants were evaluated periodically to acquire clinical information and partake in CSF biomarker investigations. Additional file The collection and processing of CSF biomarker were conducted in accordance with the PPMI biologics manual. CSF GFAP concentrations were quantified by Roche NeuroToolKit (NTK) on a cobas e 411 analyzer at Covance Greenfield Laboratories  .® electrochemiluminescence immunoassay (ECLIA) using a completely automated cobas e 601 analyzer  [CSF Aβ42, total tau (T-tau) and phosphorylated tau (P-tau) concentrations were quantified through an Elecsyszerland) . The levzerland) .Genomic DNA was extracted from the whole blood of PD subjects for processing and analysis. Apolipoprotein E (APOE) genotypes were detected by allele-specific oligonucleotide probes labeled with a fluorogenic reporter (TaqMan method) . BesidesThe cognitive assessment scales included in this study were as follows: global cognitive functioning (MoCA), episodic memory , visuospatial abilities , executive functioning/working memory , language (Semantic Fluency Test), and processing speed/attention . Cognitive status of participants was classified based on the criteria used in previous studies , 31: norp < 0.05) and hence were log10-transformed to approach the normal distribution  were utilized to assess the effect of baseline GFAP level or the interaction of baseline GFAP level with follow-up time on other measured longitudinal data. We analyzed models with tertile and continuous variables. Mediating effect analysis was used to determine whether CSF GFAP mediates between CSF Aβ42 and T-tau or P-tau. Due to the shared neurological spectrum between PD and AD , the AD-Next, the cumulative incidence of cognitive progression among various groups during follow-up was compared using Kaplan–Meier survival curves. In addition, the relationship between baseline CSF GFAP level and cognitive stage transition during follow-up was analyzed using multivariate Cox regression models.The statistical analyses in this study were conducted via SPSS 25.0. All regression analyses and GLMM were subject to adjustments for confounding variables, including age, gender, education level, APOE ɛ4 carrier status, and disease duration. In addition, to exclude the effect of CSF biomarkers on PD cognition, we also included α-syn, Aβ42 and P-tau in the analysis.The demographic and clinical characteristics of the study cohort, comprising 210 individuals with de novo PD, are presented in Table p < 0.001), except for Aβ42. In addition, statistically significant variations in age (p < 0.001) and gender (p = 0.046) were observed among the three groups.PD patients were stratified into three groups according to tertiles of baseline CSF GFAP concentrations, which exhibited analogous baseline characteristics with respect to disease duration, education level, APOE ε4 allele status, and performance on global cognitive assessment. Notably, the levels of CSF biomarkers associated with neurodegeneration varied significantly among the groups   , while no difference in CSF GFAP was observed between APOE ε4 carrier status (p = 0.384) p < 0.001p = 0.024), while no significant difference was observed in CSF GFAP levels between the A + and A − groups (p = 0.205) , T-tau , P-tau , and α-syn   or CSF P-tau (T + and T −). The T + group displayed higher levels of CSF GFAP compared to the T − group (05) Fig. . Followip = 0.874)   Fig. . Moreovep = 0.034), T-tau , and P-tau , α-syn   , but also in episodic memory , visuospatial abilities , language , as well as processing speed/attention  , the higher the baseline CSF GFAP level of the participants, the higher the risk of cognitive transition . However, in the PD–MCI group (n = 68), no significant predictive effect of CSF GFAP on the risk of dementia was found (p = 0.597). Meanwhile, multivariate Cox regression analysis revealed that higher baseline CSF GFAP levels in PD–NC patients could still predict the risk of dementia during 8 years of follow-up, even after controlling for age, gender, education level, APOE ε4 carrying status and disease duration  in patients with newly diagnosed PD over an 8-year follow-up period, we performed Kaplan–Meier survival curves and Cox regression analysis. Figure Finally, to exclude the influence of other CSF biomarkers on PD cognition, we included several extra confounding factors , yielding results similar to those presented above  and the cognitively impaired group (MCI and AD dementia) . We founFerrari-Souza et al. demonstrated that CSF GFAP levels were influenced by Aβ pathology in aging and AD. Then, they discovered that CSF GFAP partially mediated the impacts of Aβ pathology on both hippocampal atrophy and cognitive deficits through mediation analyses . Our resCertainly, an additional noteworthy aspect to be highlighted in this investigation is that through subgroup analysis considering age, gender, clinical cognitive diagnosis, APOE ε4 allele, and Aβ status, respectively, we discovered that the predictive value of GFAP baseline level on cognitive decline in PD was significant but there were subtle differences, which were manifested in different cognitive domains as well as cognitive severity. Further investigation is necessary to examine the correlations between these variables and CSF GFAP with longitudinal cognitive deterioration in new-onset PD patients.Likewise, Bartl et al. also analyzed data from PPMI. Unfortunately, they did not confirm GFAP as a prognostic biomarker for future cognitive decline and CSF biomarker changes in PD . The prin = 31) with a disease duration exceeding 1 year, for a period of 4.1 years. They discovered that baseline plasma GFAP concentrations were significantly higher in the MCI conversion group (PD patients who developed from MCI to dementia during the entire follow-up period) than in the MCI stable group (PD patients who did not develop from MCI to dementia during the entire follow-up period). Moreover, the receiver operating characteristic curve displayed that baseline plasma GFAP had a sensitivity of 90% and a specificity of 81% to distinguish MCI stable group from MCI converted group at the optimal cutoff value of 100.2 pg/ml. Taken together, these findings imply that plasma GFAP holds promise as a viable biomarker in prognosticating the advancement to dementia in PD–MCI patients [In addition, Tang et al. conducted a longitudinal study on PD–MCI patients  on longitudinal CSF biomarkers and cognitive progression. Table S4. Effects of baseline GFAP on longitudinal CSF biomarkers and cognitive progression in the tertiles. Table S5. Effects of baseline GFAP*time  on longitudinal CSF biomarkers and cognitive progression. Table S6. Effects of baseline GFAP*time on longitudinal CSF biomarkers and cognitive progression in the tertiles. Table S7. Progression risk from NC to MCI or dementia or from MCI to dementia. Table S8. Prediction of baseline GFAP and GFAP*time in male and female patients with de novo PD. Table S9. Prediction of baseline GFAP and GFAP*time in de novo PD patients aged < 56 and ≥ 65. Table S10. Prediction of baseline GFAP and GFAP*time in patients with PD–NC and PD–MCI. Table S11. Prediction of baseline GFAP and GFAP*time in new-onset PD patients carrying APOE ε4 or not. Table S12. Prediction of baseline GFAP and GFAP*time in patients with Amyloid-PD and Amyloid + PD.


PTPN11 (HGNC:9644) Exon 3 that contains 30 different PTPN11 Noonan syndrome (NS) mutation sites. We found 14 of these variants formed clusters among the testes; one testis had 11 different variant clusters. The mutation frequencies of these different clusters were not correlated with their case‐recurrence rates nor were case recurrence rates of PTPN11 variants correlated with their tyrosine phosphatase levels thereby confusing PTPN11's role in germline clonal expansion. Six of the PTPN11 exon 3 de novo variants associated with somatic mutation‐induced sporadic cancers (but not NS) also formed testis clusters. Further, three of these six variants were observed among fetuses that underwent prenatal ultrasound screening for NS‐like features. Mathematical modeling showed that germline selection can explain both the mutation clusters and the high incidence of NS (1/1000–1/2500).Some spontaneous germline gain‐of‐function mutations promote spermatogonial stem cell clonal expansion and disproportionate variant sperm production leading to unexpectedly high transmission rates for some human genetic conditions. To measure the frequency and spatial distribution of de novo mutations we divided three testes into 192 pieces each and used error‐corrected deep‐sequencing on each piece. We focused on  PTPN11 variants show spatial clustering in the testis due to germline selection explaining the high birth incidence of Noonan syndrome. Some somatic PTPN11 cancer mutations are similarly clustered in testis.Mutation frequency heat map of spontaneous  Examples of genes that acquire such deleterious RAMP/PAE variants are FGFR2 (HGNC:3689), FGFR3 (HGNC:3690), RET (HGNC:9967), HRAS (HGNC:5173) and PTPN11 (HGNC:9644) , a common Mendelian genetic condition occurring once in every 1000–2500 births  of NS results from mutations  lists 110 different annotated NS1 missense variant sites reported in the 593 amino acid coding region of SHP‐2. NSEuroNet reflects an earlier compilation of NS1 variants . E3 spans amino acids 46–82 in SHP‐2 and contains 108 coding nucleotides including 30 NS1 sites  with varying case recurrence rates  in the general population that typically arise only from somatic mutations . We found germline testis clusters carrying sporadic‐only (S‐O) cancer variants  and (RefSeq NP_002825.3), respectively. All the variants notations are consistent with HGVS nomenclature using VariantValidator . Throughout the paper all NS1 base substitutions, for example the variant NM_002834.5:c.188A>G, is listed as c.188A>G and the resulting amino acid change NP_002825.3:p.(Tyr63Cys) as p.(Tyr63Cys).The Accession and Version numbers for the PTPN11 disorder NSML . The donors  had not been treated with drugs that interfere with normal spermatogenesis and their testes were frozen 10–12 h after death and stored at −80°C.2.1.2One testis from each unaffected donor was divided into approximately equal pieces . Details of the testis dissections, DNA isolation and quantitation of the amount of DNA in each piece have been published previously  to the PCR primers used in the first two amplification rounds .A total of 576 libraries (3 donor's testes × 192 pieces from each testis) were constructed using the SSS Sequencing System . The average number of UID families per testis piece is 64,000. The number of UID families per testis piece is listed in Data For each testis piece, we determined the variant frequency for each of the 127 consecutive E3 nucleotide sites (108 coding sites and 19 intron sites × 3 possible variants per site = 381) by dividing the number of different UID families with the same base substitution by the total number of different UID families captured from that piece. The precision with which we can measure a mutation frequency per testis piece is one divided by the number of different UID families in that piece. Data −6 for four of the six possible variant types, and 5–6× higher for the remaining two types (G>T/C>A and C>T/G>A) likely due, respectively, to oxidation and deamination damage associated with PCR during the first two SSS cycles. Using the 21‐year‐old testis as a control (this testis does not exhibit mutation clusters), Figure Previously , but three of the pieces in this testis are colored red or orange indicating a variant frequency greater than 10−3. The highest frequency in all of this testis’ pieces is 4.8 × 10−3 . The average c.182A>G variant frequency in this donor's testis, calculated using all the testis pieces is 1.7 × 10−5 (175 distinct mutant c.182A>G UID families among the 10 million different UID families identified in this testis). The variants are not uniformly distributed spatially as one would expect if the SSC mutation rate per cell division were elevated at the c.182A site but are clustered indicating a selective advantage for those cells carrying the variant  database. All of the testis pieces are as expected and colored a shade of gray with no testis pieces with high variant frequencies. For example, the observed average testis frequency for c.183A>T is 1.0 × 10−7 (1 mutant UID family out of the 10 million families detected in this testis), more than two orders of magnitude lower than the testis average for the c.182A>G variant.In contrast, Figure 3.1.1Data We defined a significant de novo variant cluster as those instances in which the observed MaxPF was exceptionally high relative to the average testis frequency. We defined “exceptionally high” based on computer simulations of the neutral (without selection) germline model of cell proliferation and mutation from zygote formation through adulthood .Figure The 21‐year‐old testis, unlike the two older testes, had no variants with high MaxPFs Figure , confirmThe two testes from the older unaffected donors captured 47% of the 30 known E3 NS1 variant sites ; the average number of cases for these sites is 2.9  between the average testis variant frequency and the number of reported NS1 cases.The chance that a man passes a new mutation to his child is presumably the average testis frequency for this variant at the age of fatherhood. Figure 3.1.3PTPN11 contains E3 nucleotide sites that can undergo somatic missense mutations causing sporadic cancers in the general population . Figure 3.1.4To emphasize that different E3 variant sites can form clusters that grow independently in the same testes, Figure 3.1.5Of the 660 possible E3 variants not found to cause either NS1 or cancer, only two are significantly clustered. One is a synonymous variant c.216C>T p.(Ala72Ala) in the 68‐year‐old donor. A de novo synonymous variant arising in a single SSC already carrying a de novo nonsynonymous mutation with a selective advantage could form a significant testis cluster containing both variants  databases for the presumptive NM_002834.5:c.A>G (PTPN11 12:112450344A>G) variant c.164A>G p.(Lys55Arg) without finding it. Importantly, the Genome Aggregation Database  did not contain p.(Lys55Arg) among a total of 250,000 sequenced alleles that included approximately 109,000 sequenced “control” alleles.The NS1 p.(Lys55) codon within the 97 amino acid N‐terminal Src homology 2 domain (N‐SH2) plays a critical role in SHP‐2 function 's likely functional impact. PolyPhen‐2  whether the recurrence rate for cases at any particular NS1 variant site depend on the amino acid substitutions effect on SHP‐2's phosphatase activity and (2) if germline selection of many PNPN11 variants we first demonstrated that the ratio of transitions to transversions (Ti/Tv) in mutations causing NS1 is the same in E3 as in the rest of PTPN11 .To justify combining our E3 data with other NS1 3.1.8PTPN11  and shared very similar substrates and experimental designs. Each data set used a basal substrate alone or the basal substrate plus a peptide containing a phosphotyrosine known to bind SHP‐2; the two datasets used different basal substrates and phosphopeptides. We evaluated 12 SHP‐2 NS1 variant sites (four from E3) ranging from 7 to 241 reported cases/site; average cases/site = 53. Figure p value 0.24, correlation −0.21). In addition, SHP‐2 phosphatase activity of the three tested PTPN11 S‐O cancer variants, shown in Figure p value <3 × 10−5, explained in Methods We originally speculated the varying NS1 recurrence levels might be correlated with the extent of SHP‐2 tyrosine phosphatase hyperactivation characteristic of the different NS1 amino acid changes. Since higher levels of hyperactivation are generally associated with poor developmental outcomes Kratz, , 2006 we3.1.9−4 which is in good agreement with the observed NS birth rate estimate range (4 × 10−4–10−3). Details in Methods Earlier studies could explain testis clusters by a germline selection model . In Methods RAMP/PAE mutations in receptor tyrosine kinases (RTK) have been shown to have significant mutation clusters caused by germline selection , a PTPN11‐driven sporadic blood cell cancer case may result in the general population whereas if the same nucleotide substitution arises in an SSC the variant may be transmitted to the next generation  variant cluster we found requires studies on the functional properties of a synthetic version of this presumptive variant to decide whether it is a rare but unlikely neutral variant cluster  allowed us to predict a NS birth rate that is within the generally accepted range (1/1000–1/2500 births) ClinVar DECIPHER, DatabasE of genomiC varIation and Phenotype in Humans using Ensembl Resources (https://ensembl.org/index.htmlE!Ensembl https://nseuronet.comEuropean Network on Noonan syndrome and related disorders database https://gnomad.broadinstitute.org/gnomAD (v2.1.1), https://VariantValidator.org)HGVS nomenclature using VariantValidator (https://www.omim.orgMendelian Inheritance in Man (MIM#) http://www.ncbi.nlm.nih.gov/bioproject/NCBI BioProject database (http://genetics.bwh.harvard.edu/pph2/dbsearch.shtml),PolyPhen‐2 (http://structure.bmc.lu.se/PON-P2/)PON‐P2 Protein Variation Effect Analyzer (http://provean.jcvi.org/index.php),PROVEAN (https://www.ncbi.nlm.nih.gov/protein/33356177PTPN11 https://www.uniprot.org/uniprot/Q06124SHP‐2 https://www.uniprot.org/uniprot/P40763STAT3 UniProtKB ‐ P40763 (STAT3_HUMAN) Experiments were designed by J.E., P.C and N.A and carried out by J.E. using methods developed by S‐R.Y, S‐K.C. and J.E. Both A.S and V.K contributed to SHP‐2 protein structure‐function analysis. P.C was responsible for sequencing data and statistical analyses. The manuscript was written by N.A. and P.C. who both had overall supervision and conceived of the project.The authors declare no conflicts of interest.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


The high data quality and phase sensitivity of the technique have allowed the reconstruction of all four phases namely pore space, IrO2, TiO2 support matrix and the ionomer network, the latter of which has proven to be a challenge in the past. Results show that the IrO2 forms thin nanoporous shells around the TiO2 particles and that the ionomer has a non-uniform thickness and partially covers the catalyst. The TiO2 particles do not form a percolating network while all other phases have high connectivity. The analysis of the CL ionic and electronic conductivity shows that for a dry CL, the ionic conductivity is orders of magnitudes lower than the electronic conductivity. Varying the electronic conductivity of the support phase by simulations, reveals that the conductivity of the support does not have a considerable impact on the overall CL electrical conductivity.Reducing precious metal loading in the anodic catalyst layer (CL) is indispensable for lowering capital costs and enabling the widespread adoption of polymer electrolyte water electrolysis. This work presents the first three-dimensional reconstruction of a TiO However, PEWE is characterized by high operational and capital expenditures which combined with the scarcity of platinum group metal based electrode materials , currently limit the widespread commercialization of the technology2.Polymer electrolyte water electrolysis (PEWE) is considered a key technology for producing green hydrogen using renewable energies. PEWE offers high current density, high hydrogen purity and excellent dynamic response to intermittent power fluctuations, making it ideal for meeting the intermittent demands of solar or wind power4 until it reaches the anodic CL where the oxygen evolution reaction (OER) takes place. The corrosive acidic environment provided by the ionomer membrane (pH ∼ 0) and the high cell operating potential (> 2 V), impose the use of scarce and expensive catalyst materials, often platinum group metals such Ir or Ru. This not only increases the cost of the electrolyzer  but, due to the limited annual production of Ir, also limits the installable power capacity to ~ 2 GW a-11. It is therefore essential to optimize the anodic catalyst layer, to reduce catalyst loading while maintaining high cell current density and efficiency.A typical PEWE cell is composed of two catalyst layers (CLs) that serve as the electrodes, which are deposited onto a polymer electrolyte membrane (PEM). Liquid water is fed from a flow field at the anode side and distributed through a titanium porous transport layer (PTL)2-based catalysts are among the most commonly employed materials due to their relatively high activity and long-term stability for the oxygen evolution reaction (OER)5. The employment of a support promotes the dispersion of the IrO2 nanoparticles, increasing the electrochemically active surface area and thereby reducing IrO2 loading. Titania (TiO2) is the most commonly used support because of its high electrochemical stability and low cost5.Despite the high costs and limited availability, IrO2 particles catalyze the OER reaction and are responsible for the CL electrical conductivity. The ionomer acts as a binder and provides ionic transport of the produced protons. Finally, the pores in the CL ensure the two-phase transport of water and oxygen to and from the reaction sites.A typical anodic CL is usually coated onto the electrolyte membrane, using an ink composed of the catalyst powder mixed with a proton conducting polymer (ionomer). Upon drying, the mixture forms a complex three-dimensional microstructure. For the water-splitting reaction to occur, electrons, protons, and water/oxygen need to be transported efficiently in the respective phases. The IrO2, TiO2) and respective networks is essential for future, rational CL designs aiming at low loading and high efficiency. The challenge of anodic CL microstructure analysis lies in the difficulties of achieving high-quality 3D reconstructions, considering the small feature size (< 1 µm6). While three-dimensional reconstructions of PTLs can be easily found in literature for both ex situ7 and operando studies8, only one recent publication can be found where a PEWE CL reconstruction was obtained6. In6, the authors used focused ion beam-scanning electron microscopy (FIB-SEM) to reconstruct a CL employing an unsupported Ir-Ru catalyst. However, the ionomer structure could not be resolved and the transport properties of the electrode were deduced by artificially introducing the ionomer phase. Furthermore, the reconstruction of the ionomer phase requires the use of transmission electron microscopic tomography9 but this results in a reduction in the domain volume, analyzed for the higher resolution. In the last decade, X-ray ptychographic computed tomography (PXCT) was proven effective for reconstructing the microstructure of porous media for many energy devices, from batteries11 to solid oxide cells14. PXCT offers a unique combination of high spatial resolution and high sensitivity to differences in material density15 making it ideal for the analysis of the multi-phase microstructure of the PEWE CL.Therefore, the characterization of all four phases  and a Pt on carbon hydrogen evolution reaction (HER) catalyst  were used. Both IrO2 and TiO2 are in the rutile form, as shown by Pătru et al.17.The samples used for the PXCT procedure were extracted from representative catalyst coated membranes (CCMs), routinely used for electrochemical characterizations2-TiO2 powder. The concentration of the Nafion solution was 5 wt. %. The ink mixture was homogenized for 45 min. A Nafion® membrane N115  was employed. The Nafion ionomer content relative to the total weight of the catalyst was set to 11 wt%, as optimized by Bernt et al.18 for IrO2 on TiO2 catalysts.The anode catalyst ink was prepared by stepwise addition of ultrapure water, 2-propanol, and Nafion solution  to the IrOIrO2 cm-2 was achieved by monitoring the online loading based on a reference sheet. Subsequently, the CCMs were dried for 12 h under ambient conditions. An in-depth description of the automated spray coating process is reported in16.The spraying procedure was performed using an automated benchtop coating system . The anodic target loading of 3 mg19, performed using the Ga-ion beam of a focused-ion-beam/scanning electron microscope . The sample was extracted using a micro-manipulator in the FIB-SEM and transferred onto the OMNY pin20 for mounting on the beamline rotation stage. More details on the lift-out procedure can be found in Section From the anodic CL, a pillar with a quasi-octagonal cross-section was extracted using the lift-out technique21, at cryogenic temperature (90 K) and under vacuum (2 × 10–8 mbar).The PXCT measurement was performed at the X12SA (cSAXS) beamline at the Swiss Light Source, Paul Scherrer Institut, Switzerland. To avoid potential beam damage and reduce the mechanical degradation of the ionomer, the experiment was carried out using the OMNY microscope2. The scanning points follow a Fermat spiral trajectory with average 1.5 μm step size. At each scanning position, a coherent diffraction pattern was recorded 7.2 m downstream of the object using an in-vacuum Eiger detector with 75 × 75 μm2 pixel size at 0.1 s exposure time. The illumination was defined by a gold Fresnel zone plate with 60 nm outermost zone width and a diameter of 220 μm, with the sample placed 2.4 mm downstream from the focal spot. Diffraction patterns with sizes of 1000 × 1000 pixels were used in the ptychographic reconstruction, giving an object pixel size of 16.4 nm. The ptychography reconstructions were carried out using the difference-map algorithm with a maximum likelihood refinement step22 using the PtychoShelves package23. For the tomogram, 1550 projections equally spaced over an angular range of 180° were collected. Three-dimensional ptychographic reconstruction were obtained through post-processing and fine alignment of the reconstructed 2D projections and a modified filtered back projection algorithm24.Ptychographic projections were measured at 7.2 keV photon energy, each with a scanning field of view of 19 × 11 μm13. The particle size distribution (PSD) was calculated from the segmented data using the continuous particle size distribution method25. The interface analysis between the different phases is performed as in26. The segmentation, interface analysis and PSD are computed using in-house Matlab and Python scripts. All plots were produced in MatLab 2020b while the 3D renderings in Fig. The three-dimensional dataset was segmented using a 2D histogram thresholding procedure, which involves the determination of segmentation thresholds based on the intensity and the magnitude of the intensity gradient, as described in27. Thus, every mesh element had the same dimensions as the voxel resolution for the microstructure. The meshes were generated using the open-source library pyfcst which is a sub-library of the open-source package OpenFCST28.To perform numerical simulations on the catalyst layer microstructure, a voxel-based mesh was generated by direct conversion of the microstructure voxels to mesh elementsThe numerical simulations to compute the tortuosity and two-phase effective conductivity were performed using the open-source package OpenFCST. To compute the tortuosity for the different phases, a Laplace equation of the form:entclass1pt{minimaThe tortuosity  effective electronic conductivity, modified meshes consisting of both phases with different material ids were generated. For the numerical simulations, the coefficient 3 is extracted from the entire tomogram. Figure For the analysis of the electron density and phase fractions, a sub-volume of ~ 9 × 9x7 µm29, we measure a half-pitch resolution of ~ 16.7 nm, close to the employed voxel size. Details of the FSC calculations can be found in section Using the three-dimensional Fourier Shell Correlation curve with a half-bit threshold (FSC)30. Away from the absorption edges of the constituent materials, the 3D electron density distribution 31.After tomographic reconstruction, PXCT provides the three-dimensional distribution of the complex-valued refractive indexFigure 2 particles are mostly dense, presenting a negligible internal porosity.As expected, the values related to the porosity are centered around zero, the negative electron density values are due to statistical fluctuation related to the image noise. The expected mass density of the titanium(IV) oxide phase, in the rutile form, lies within the interval of values obtained experimentally from ptychography, testifying that the TiO332. Considering that the ionomer employed has an equivalent weight 32, the value calculated from the tomogram is very close to that of a bulk Nafion membrane. While it was hypothesized that the ionomer mass density can depend on the film thickness33, results suggest that the ionomer in the CL, for films with a thickness greater than ~ 17 nm, presents the same mass density as in the bulk membrane.In dry conditions, the commonly reported value for the density of dry Nafion membranes is around 2.1 g/cm2 phase than the theoretical value. This suggests the presence of nano-porosity within the IrO2 particles that are not resolved using PXCT. These nano-pores lead to the apparent reduced mass density and also contribute to additional surface roughness which cannot be resolved with the present resolution of 16.7 nm. Considering the ratio between the IrO2 theoretical and measured mass density and assuming that the nanopores are empty, we can estimate a 25% porosity for the IrO2 phase. However, this estimate might vary considerably in case the internal IrO2 porosity is partially or fully filled with the ionomer phase. Further analyses using more sensitive techniques such as mercury intrusion porosimetry (MIP) or TEM tomography will be required to clarify this point.However, a considerably lower mass density is measured for the IrO2, TiO2, ionomer, and pores are calculated from the segmented data, using the biggest rectangular volume (~ 9 ×  9 × 7 µm3) extractable from the quasi-cylindrical pillar. A representative elementary volume (REV) analysis has been performed to ensure reliability of reported values. For the analysis, 50 sub-volumes of varying sizes have been sampled from the entire dataset from random locations. For each sub-volume set, the average value of the phase fraction and its standard deviation have been computed. The results of the representativeness analysis are presented in Fig. The volume fractions of IrO3, the relative standard deviation ranges from a minimum of less than 1% (Pores) to a maximum of 7% (TiO2) suggesting that the volume investigated can be considered representative for further analysis.Figure 2 and TiO2 can be deduced from the catalyst loading employed. The average thickness of the catalyst layer was determined by averaging the distance between the Nafion membrane and the carbon coating (electrode surface), as explained in section 3, determined assuming that the catalyst consists of iridium(IV) oxide and titanium(IV) oxide as follows: Knowing the weight percentages of the components (86.9 wt% IrO2 and 13.1 wt% TiO2) and the density of the materials (11.7 g/cm3 for IrO2 and 4.23 g/cm3 for TiO2), we can calculate the IrO2 and TiO2 volume fraction of 27% and 11% respectively. Similarly, the same equation can be applied to calculate the ionomer volume fraction:The phase fractions in the electrode can be compared to the theoretical volume fractions computed from the ink composition (used for the spray coating procedure), the density of the constituent materials, and the thickness of the catalyst layer. The volume fractions for the IrO2 and pore phase fraction corresponds quite well with the expected values based on the ink composition. However, from the tomographic data, the IrO2 volume fraction is overestimated while the measured ionomer phase fraction is considerably lower than the one expected.Table 2 volume fraction is likely due to the hypothesized internal nano-porosity, which is neglected in the segmentation, resulting in a higher IrO2 volume fraction value. Similarly, the discrepancy observed for the ionomer phase fraction could be also explained assuming that not all ionomer phase is detected. The ionomer could be present as a thin film (< 10 nm thick) covering the surface of the catalyst particles, in this form it would not be detected by the segmentation. Furthermore, taking into account the nano-porosity hypothesized for the IrO2 phase, the ionomer could be present in between the nanoparticles.The overestimation of the IrO2/TiO2 particles. Support particles (TiO2) present a wide range of diameters (0–800 nm), with a peak around 300 nm  shares its surface only with the IrO2 phase and therefore is not reported in the calculations. From the tomograms, the values for the different interface and surface areas are shown in Table The microstructure of the catalyst layer determines the accessibility of the catalyst and is crucial for electrochemical reactions. The active area is usually referred to as the electrochemically active surface area (ECSA) and it is an important parameter for determining the overall activity of the catalyst layer, hence influencing the PEWE cell performance. In the tomographic data, the support phase  at the surface of the catalyst layer seems probable, as this would be in line with the missing ionomer volume fraction observed  might be covering the IrO2 surface, the uneven ionomer distribution achieved using spray coating might also result in only partial coverage of the IrO2 surface by the ionomer. Higher-resolution imaging techniques such as TEM tomography, could provide further insights into the thin film ionomer distribution on the IrO2 surface and provide a more accurate estimate of the IrO2/Ionomer interface area.Figure In the catalyst layer, reactants and products have to be transported to and from the active sites. The morphology of the microstructure is crucial for the transport of each species. Table 2) is 0, testifying that the TiO2 particles are fully isolated. The catalyst and pore phase present almost complete connectivity. Interestingly, the ionomer phase also has a high connectivity (95%), despite the smaller phase fraction when compared to the IrO2 and pore phase.The connectivity of the support phase . This observation is in agreement with Bernt et al.2 percolating network, leading to a decrease in overall electronic conductivity. This effect was already hypothesized in36 and will be examined in a future in-situ tomography work. Furthermore, imperfections in the segmentation due to limits in resolution might lead to errors in the reported conductivity values. For example, the hypothesized porosity in the IrO2 is neglected, leading to an overestimation of the CL electronic conductivity. However, the good agreement with literature data36 hints that this effect has a small impact on the simulation results.However, in presence of liquid water, the expansion and deformation of the ionomer might lead to the disruption of the IrO2 nanoparticles and reduce the agglomeration of the active catalyst. However, due to the acidic environment and the high anodic potential, only some oxides or carbides offer the required stability for long-term operation. With the aim of finding the most conducting support while preserving the required chemical stability, various materials have been researched such as tantalum-doped TiO237, niobium-doped TiO238, or antimony-doped SnO2 (ATO)39.In PEWE, to reduce the precious metal loading, high surface area supports are normally used. Support materials promote the dispersion of the IrO2 connectivity, and overall CL morphology. For example, Polonsky et al.40 have reported that the support conductivity is not critical and non-conductive materials might also be considered as a support for IrO2 catalyst.However, the influence of the support conductivity on a practical CL is still unclear and depends on parameters like Ir loading, IrO2/TiO2 morphology, we can calculate the catalyst layer's overall electrical conductivity. Figure 2 intrinsic apparent conductivity5. The y-axis shows the ratio between the computed effective CL conductivity and the IrO2 bulk conductivity.Having access to three-dimensional data and spatial information on the IrO2 and tantalum-doped TiO237, ATO41 and IrO2 are indicated in Fig. 2, the range of conductivity values indicated by the green rectangle corresponds to the different levels of Ta doping, with x going from 0.05 to 0.3.In this study, the support conductivity is varied across eight orders of magnitudes, to simulate the effect of employing some of the previously reported support materials on the overall CL conductivity. As examples, the values of Overall, changing the support connectivity of eight orders of magnitudes results only in doubling the electric CL connectivity.–1 and presents a steep increase for values of support connectivity exceeding the one of IrO2The curve is relatively flat for ratios below 102 catalyst would have only a ~ 30% higher electrical conductivity than the case with the support.A pure IrOUsing the majority of the support materials reported in literature, the conductivity of CL will only be marginally affected.From Fig. 2 is ensured, any high-surface area support material can be employed, even if fully electrically insulating. Furthermore, the use of the support is always advised since the benefits in reducing Ir loading and potentially increasing the CL active area greatly outweigh the marginally reduced conductivity. Finally, even at high loading and with a full percolating IrO2, the CL conductivity can be improved only using materials with a higher conductivity compared to the IrO2.Generalizing from the catalyst materials and structure morphology employed in this work, for any CL where the percolation of the IrO2 percolating network might not exist.The importance of the support conductivity will be more pronounced for CLs with ultra-low loadings where a fully connected IrO2-supported IrO2 using high-resolution ptychographic tomography. With the unique capabilities of the technique, including the high spatial resolution, low noise, and high sensitivity to small variations in electron density, all phases  were clearly resolved.In order to characterize the catalyst layer morphology, we employed the first three-dimensional reconstruction of the OER catalyst TiO2 particles are dense and they are present in the rutile form. The mass density of the ionomer  is confirmed to be close to the bulk Nafion properties. The IrO2 phase presents an electron density lower than the expected value, indicating that the IrO2 likely has a nanoporosity below the limit of the spatial resolution of ptychographic tomography, as shown by the SEM analysis.From the analysis of the electron density, we estimated that the TiO3 is proved to be representative for all phases. The phase fraction calculations present a discrepancy to the expected values derived from the catalyst layer ink composition. The overestimation of segmented IrO2 volume fraction is caused by the presence of nanoporous IrO2 as confirmed by SEM images. The analysis of the continuous pore and particle size distribution reveals that the void phase presents a broader size distribution compared to the other phases.The analyzed volume of 9 × 9x7 µm2 but also interconnects catalyst agglomerates.For an ionomer to catalyst content of 11 wt%, the majority of the ionomer phase presents a thickness between 100 and 300 nm, however, agglomerates larger than 400 nm are also present, showing that the ionomer does not form a uniform layer covering the surface of the IrO2/g, a factor of ~ 25 lower than reported in literature, testifying that the IrO2 nanoscale surface roughness presents a rugosity below the available spatial resolution of 16.7 nm. Furthermore, in the segmented volume, only ~ 53% of the IrO2 surface appears covered by the ionomer with a layer thickness of > 16 nm, hinting at the possibility that the rest of the catalyst surface is not fully covered by the ionic conductive phase or covered by a thinner layer.The analysis of the phases’ interface areas reveals a total catalyst surface of 1.2 mResults show that the pore phase has the least tortuosity while the ionomer with 7.1 presents the highest values. The effective conductivities computed from the tomographic data are comparable with previous experimentally reported values and indicate that, in the structure with a non-swollen ionomer, the ionic conductivity is the limiting component, even if the ionomer is in a well-conducting state.2 loadings, and IrO2/support ratios, the analysis of the support conductivity shows that the presence of the support phase only marginally affects the overall CL conductivity. A pure IrO2 catalyst with the same morphology possesses only a ~ 30% higher electrical conductivity. Furthermore, varying the support conductivity below the IrO2 value has only a negligible impact on the overall CL conductivity, proving that even electrically-insulating support materials can be used. Finally, for a catalyst structure with low loading and a non-percolating IrO2 phase, a support material with electrical conductivity in the range of IrO2 is required to obtain similar CL conductivities.For catalyst layers having similar morphology, IrOThis study provided not only insights into state-of-the-art catalyst layers via synchrotron-based PXCT and guidance of rational catalyst layer design but also provides a novel tool for analysis of future generation low-loaded CL microstructures.Supplementary Information.


Conclusions: Increases in NIIA and SIIA in PWH were associated with corresponding increases in ND, suggesting that reducing neuro/systemic inflammation might slow or reverse neurodegeneration.Background: HIV infection causes neuroinflammation and immune activation (NIIA) and systemic inflammation and immune activation (SIIA), which in turn drive neurodegeneration (ND). Cross-sectionally, higher levels of NIIA biomarkers correlate with increased biomarkers of ND. A more convincing confirmation would be a longitudinal demonstration. Methods: PWH in the US multisite CHARTER Aging project were assessed at a baseline visit and after 12 years using standardized evaluations. We measured a panel of 14 biomarkers of NIIA, SIIA, and ND in plasma and CSF at two time points and calculated changes from baseline to the 12-year visit. Factor analysis yielded simplified indices of NIIA, SIIA, and ND. Results: The CSF NIIA factor analysis yielded Factor1 loading on soluble tumor necrosis factor type-2 (sTNFR-II) and neopterin, and Factor2, loading on MCP1, soluble CD14, and IL-6. The SIIA factor analysis yielded Factor1 loading on CRP, D-dimer, and Neopterin; Factor2 loading on sTNFR-II. The ND analysis yielded Factor1 loading on Phosphorylated tau (p-tau) and Aβ42; Factor2 loading on NFL. NIIA Factor1, but not Factor2, correlated with increases in CSF NFL (r = 0.370,  The human immunodeficiency virus (HIV) targets and systematically undermines the immune system’s vital components, thereby compromising the host’s ability to mount effective defenses against various pathogens . Over 40The virus infiltrates the brain in the initial stages of primary infection, within the initial 14 days . Even atHIV gene products, such as gp120 and Nef, stimulate the activation of lymphocytes and macrophages, resulting in the secretion of proinflammatory cytokines and chemokines . HIV proNeurodegeneration is often assessed by measuring biomarkers of neuronal injury in plasma and CSF. The most frequently used biomarker of neurodegeneration is the neurofilament light protein (NFL), a major structural element of large-caliber myelinated axons. CSF NFL concentrations represent a sensitive marker of damage to both central and peripheral neurons in several neurologic disorders ,30,31,32A comprehensive exploration of these neurological consequences is essential not only to deepen our understanding of the virus’s multifaceted impact but also to inform the development of integrated therapeutic strategies that address both immunological and neurological dimensions of HIV infection. We measured changes in biomarker levels from baseline to follow-up over 12 years, hypothesizing that increases in inflammation would correlate with increases in neurodegeneration biomarkers. We also expected that viral suppression between the baseline and follow-up visits would be associated with decreasing markers of neuroinflammation and immune activation (NIIA) and systemic inflammation and immune activation (SIIA).PWH in the US multisite CNS HIV Antiretroviral Effects Research (CHARTER) Aging project were assessed at a baseline visit and again after 12 years using standardized evaluations. Baseline visits took place from 2003–2007; follow-up visits were performed between 2016 and 2019. Participants with severely confounding medical and neuropsychiatric conditions such as active psychosis were excluded. All participants signed informed consent documents approved by the local Institutional Review Boards at each site.Briefly, the standardized assessment included a medical history, psychiatric interviews to obtain Diagnostic and Statistical Manual Version IV (DSM-IV) diagnoses, neurological and physical examinations, and laboratory assessment as previously described . ParticiDemographics and clinical characteristics were summarized using numbers and percentages, means and standard deviations, and medians and interquartile ranges (IQR). For each biomarker available, we calculated the difference between its levels at the baseline and follow-up visit. Factor analyses were used to construct simplified indices of biomarker changes separately for biomarkers of SIIA , NIIA , and ND . Correlations of SIIA and NIIA with ND indices were evaluated using Pearson’s r or Spearman’s rho as appropriate. Follow-up analyses assessed correlations between the individual biomarkers separately. Assessment of potential confounds including demographics and indicators of HIV disease status was done using multiple regression.Participants were 108 ART-treated PWH, all virally suppressed at follow-up, with demographics and HIV disease characteristics as shown in Baseline visits occurred between September 2003 and September 2008; follow-up visits occurred between March 2016 and June 2019. The mean duration between visits was 12.5 ± 0.766 years. Participants were divided into two subgroups based on changes in viral loads between the baseline and follow-up visits: those who remained suppressed and those who became suppressed. All 55 participants who remained suppressed were on ART at baseline; 28 out of 53 PWH who became suppressed were off ART at baseline and started ART between the two visits.The SIIA factor analysis yielded two factors: Factor1 loaded on CRP, D-dimer, and neopterin; Factor2 loaded on sTNFR-II. The NIIA factor analysis yielded two factors: Factor1 loaded on sTNFR-II and neopterin, and Factor2 loaded on MCP-1, sCD14, and IL6. The ND analysis yielded two factors: Factor1 loaded on Aβ42 and p tau; Factor2 loaded on NFL.p = 0.0002). Because viral suppression is an important determinant of inflammation in PWH, we examined the interaction of CSF Immune Factor1 with viral suppression. In a multivariable model predicting CSF neurodegeneration Factor2 from viral suppression, CSF Immune Factor1, and their interaction, the interaction showed a trend towards significance (p = 0.0508); the main effect of CSF Immune Factor1 was significant (p = 0.0005), while that of viral suppression was not (p = ns). The correlation was much stronger for those who remained suppressed  than for those who became suppressed .p = 0.023), as can be appreciated in In the remained suppressed group, the proportion of females was higher than males .The time between baseline and follow-up visits was not significantly related to any biomarker change factors (p = 2.60 × 10−6) and CSF NFL . However, older age was not associated with changes in these same biomarkers.ps > 0.20). At baseline, among the 83 participants who took antiretroviral, six different regimens were used. We classified the regimens into the following groups: non-nucleoside reverse transcriptase inhibitor (NNRTI)-based (n = 1), nucleoside reverse transcriptase inhibitor (NRTI)-based (n = 2), NNRTI/NRTI (n = 33), protease inhibitor-based PI/NRTI (n = 38), PI/NNRTI (n = 2), and three-class (n = 7). At follow-up, among the 107 participants who took antiretroviral, there were nine regimens used, including integrase inhibitor (II)-based/NRTI (n = 45), NNRTI-based (n = 1), NRTI-based (n = 2), NNRTI/NRTI (n = 23), PI/NNRTI (n = 1), PI/NRTI (n = 12), PI/II (n = 1), three-class (n = 21), and four-class (n = 1). There was no significant relationship between regimen type and any CSF Immune or CSF Neurodegeneration change Factors . Nonetheless, we excluded groups with fewer than eight patients, and even this exclusion revealed no significant association between regimen type and changes in CSF Immune or CSF Neurodegeneration factors.Sex, ethnicity, and BMI did not significantly influence any of the CSF Immune or CSF Neurodegeneration change Factors , while those who became suppressed had significant decreases in CSF Immune Factor1 . The difference between the two subgroups was statistically significant (p = 0.0022). Baseline CSF sTNFR-II was not different in those who became suppressed (2.76 ± 0.246) from those who remained suppressed . CSF neopterin was lower in those with suppressed plasma HIV RNA at baseline than in those not suppressed . Similarly, CSF neopterin at baseline was lower in those who had suppressed CSF HIV RNA < 50 c/mL than in those not suppressed at baseline .CSF Immune Factor1 was not related to any of the other neurodegeneration biomarkers . Those who remained virally suppressed in plasma had non-significant increases in CSF Immune Factor1 (p = 0.0327). In a multivariable regression predicting CSF Factor2 from plasma Immune Factor1, in addition to change in detectability and their interaction, only the main effect of plasma Immune Factor1 was significant .Increases in plasma Immune Factor1 , correlated with increases in CSF Neurodegenerative Factor2  neuropathogenesis. AD is a profound neurodegenerative condition and the prevalent source of dementia linked to neurodegeneration among older individuals. This disorder is marked by a gradual decline in cognitive abilities, linked to the diminishment of synaptic and neuronal components and the presence of senile plaques and neurofibrillary tangles (NFT) in the brain . There iA strength of this study is the long longitudinal follow-up and biomarker assessments at both baseline and follow-up. This helps us understand how things change over time, giving us a fuller picture. It is important to acknowledge that our study, while informative, does come with certain limitations. One notable aspect is that we did not assess all the possible biomarkers associated with inflammation and neurodegeneration. While we have made valuable strides in understanding these processes, there could be other biomarkers that we have not explored, and their inclusion might provide a more comprehensive view of the subject matter. We selected our biomarkers based on previous literature showing relationships between HIV disease and neuropathogenesis. Additionally, while most PWH are virally suppressed in modern cohorts, ours had substantial numbers of participants who were not suppressed at baseline. Nevertheless, all were suppressed at follow-up. Thus, our sample might not be representative of individuals with durable viral suppression.Our study suggests that effectively treating neuroinflammation could potentially reduce neurodegeneration in virally suppressed PWH. Anti-inflammatory medications like TNF-α inhibitors might hold promise in preventing neurodegeneration by curbing inflammation. Further research and clinical trials are needed to confirm this possibility and explore the potential of anti-inflammatory interventions for preserving neural health in this population. Another question that remains unresolved is why PWH who have remained virally suppressed still have increased neuroinflammation. Indeed, we found that sustained viral suppression was associated with longitudinal decreases in neuroinflammation. There are several theories discussing probable mechanisms in continued immune activation in PWH undergoing ART. First, neuroinflammation increases in older people despite the lack of a frank immunosuppression . Our stuIn conclusion, our study addresses the relationship between inflammation and neurodegeneration in virally suppressed people with HIV. We found that increased neuroinflammatory markers, including neopterin and sTNFR-II, were associated with elevated levels of neurodegenerative biomarkers, such as NFL, in virologically suppressed individuals. This underscores the potential impact of sustained inflammation on neural health even in the presence of viral suppression. While we did not observe significant connections between inflammation and Alzheimer’s disease markers in HIV, our findings emphasize the need for further exploration of the underlying mechanisms driving neuroinflammation in this population. The possibility of mitigating neurodegeneration through effective neuroinflammation treatment, possibly involving anti-inflammatory agents like TNF-α inhibitors, presents a promising avenue for future research and therapeutic interventions. Our results show older age was not associated with changes in these same biomarkers. As older individuals with HIV exhibit heightened neuroinflammation, systemic inflammation, and neurodegeneration, understanding these processes is crucial to developing strategies that preserve neural health and improve the well-being of people living with HIV. Further studies are warranted to unravel the complexities of neuroinflammation in virally suppressed individuals and its implications for neurological outcomes.