The morbidity and mortality of cardiovascular diseases (CVDs) have been increasing year by year all over the world and expanding greatly to the younger population, which becomes the leading causes of death globally that threatens human life safety. Prediction of the occurrence of diseases by using risk related adverse events is crucial for screening and early detection of CVDs. Thus, the discovery of new biomarkers that related to risks of CVDs are of urgent in the field. Retinol-binding protein 4 (RBP4) is a 21-kDa adipokine, mainly secreted by adipocytes. Besides its well-established function in the induction of insulin resistance, it has also been found in recent years to be closely associated with CVDs and other risk factors, such as hypertension, coronary heart disease, heart failure, obesity, and hyperlipidemia. In this review, we mainly focus on the progress of research that establishes the correlation between RBP4 and CVDs and the corresponding major risk factors in recent years. Retinol-binding protein 4 (RBP4), whose gene is located near chromosome 10 (10q23-q24), is a 21-kDa protein secreted by hepatocytes and adipocytes. RBP4, as the sole carrier of retinol in the blood, increases the hydrophilicity of retinol upon binding and completes the transport of retinol from the liver to target tissues. Retinol-bound RBP4 (HoloRBP) further complexes with the tetrameric structure of transthyretin protein (TTR) to form a retinol transport complex, which prevents glomerular filtration of HoloRBP  and effeMultiple factors have now been found to influence circulating RBP4 concentrations. Adipose tissue is not only an energy-preserving tissue but can also release numerous substances known as “adipokines” or “adipocytokines.” Adipocytes are the main source of RBP4 secretion, Atrial natriuretic peptide (ANP) directly regulates the secretory activity of adipocytes in adipose tissue  and reduThere are other factors regulate the serum RPB4. The effect of exercise on RBP4 levels depends on the intensity of exercise , with hiIn recent years RBP4 have achieved significant efficacy coronary heart disease, hypertension, heart failure . In thisAbnormal lipid metabolism is the most important risk factor for atherosclerosis, and hyperlipidemia, which includes hypercholesterolemia, hypertriglyceridemia, or both, requires binding to apolipoprotein plasm in plasma in the form of lipoproteins due to its lipid-soluble physical properties. The expression of RBP4 is negatively correlated with blood cholesterol (TC) levels . and theObesity, one of the risk factors for atherosclerosis, can lead to increased blood triglyceride and cholesterol levels. The close correlation between visceral adiposity and cardiovascular disease has been previously demonstrated . Won et Oxidative stress-mediated changes promoted the development of cardiovascular disease . It was via NADPH oxidase and NF-κB-dependent pathways  is now known to be widely used as a diagnostic indicator of heart failure. Previous studies in elderly patients hospitalized with heart failure have shown that RBP4 did not correlate with NT-proBNP, and that changes of blood RBP4 levels are more likely attributable to the deterioration of renal function in patients with advanced heart failure, resulting in the accumulation of circulating adipokines . It was Retinol-binding protein 4 is involved in the pathological process of heart failure through a variety of mechanisms. The elevation of RBP4 in patients with advanced heart failure may result from upregulation of RBP4 mRNA expression by IL-8 . In addiRetinol-binding protein 4 levels in patients with coronary artery disease were found to be significantly higher than in the non-coronary artery disease (CAD) group  and posiPreviously, Mallat found that RBP did not provide additional predictive value compared to traditional risk factors in normal subjects . But in Cardiac amyloidosis (CA) is an accumulation of insoluble fibrous deposits composed of abnormally folded protein molecules in the myocardial interstitial, which mainly manifests clinically as cardiac insufficiency, various arrhythmias, and angina pectoris, among which the transthyretin amyloidosis (ATTR) type is more common in clinical work. It was found that blood RBP4 levels in patients with transthyretin amyloidosis were significantly lower than in controls ,b. Chan In vitro assays have shown that the RBP-retinol complex (holoRBP) inhibits the rate of amyloid fibril formation in a concentration-dependent manner (Retinol-binding protein 4 has been found to have anti-amyloidogenic properties, and the decrease of RBP4 alleviate the formation of the RBP4-retinol complex and attenuates its role in stabilizing the TTR tetrameric structure . In vitrt manner . holoRBPt manner . RBP4 cot manner ; Table 1As an adipokine, RBP4 has shown a close association with dyslipidemia, obesity, and vascular impairment. RBP4 also derives exclusively from hepatocytes, but liver-secreted RBP4 does not impair glucose homeostasis . In addiXG conceived the study and designed the study protocol. YJ and JS conducted the literature review and drafted the manuscript. TS reviewed the manuscript for intellectual content, made revisions as needed. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Cryptococcal meningitis (CM) has a high morbidity and mortality due to the low detection of Cryptococcus in cerebrospinal fluid (CSF) during the early stage of the disease with traditional methods.In addition to the traditional methods of India ink staining and cryptococcal antigen (CrAg), we used nanopore sequencing and next-generation sequencing (NGS) to detect pathogenic DNA in CSF samples of three patients with CM. The CSF samples of all three patients were positive by India ink staining and CrAg. NGS also detected Cryptococcus in all three CSF samples. Nanopore sequencing detected Cryptococcus in two CSF samples.Nanopore sequencing may be useful in assisting with the clinical diagnosis of CM. Further research is needed to determine the sensitivity and specificity of nanopore sequencing of CSF. Cryptococcal meningitis (CM) is an opportunistic infectious disease of the central nervous system (CNS) with high morbidity and mortality . CM showMolecular biological methods can detect and identify pathogens rapidly and accurately without requiring culture. Polymerase chain reaction (PCR) is used in the clinical diagnosis of microbes but is limited by DNA fragment quantity. Therefore, metagenomic sequencing was used. The pathogen-positive rate of next-generation sequencing (NGS) was 28.34% in the CSF of patients with CNS infections –8. In adThis study was conducted in the People’s Hospital of Zhengzhou University. The CSF samples were collected from three patients who provided signed informed consent and stored at − 80 °C from three patients who provided signed informed consent. The samples were only used for this study.DNA was extracted from the samples with the TIANamp micro DNA kit  and sonicated to a size of 150 bp. The DNA libraries were constructed through end repair, adapter ligation, PCR amplification, and sequencing on the BGISEQ-50/MGISEQ-2000 platform. High-quality sequencing data were generated by removing short (< 35 bp), low-quality and human sequence data. The remaining data were aligned to four microbial genome databases.DNA was extracted by a QIAamp DNA mini kit . The DNA libraries were constructed using a rapid PCR barcoding kit (SQK-RPB004) and sequenced on Flow Cell R9.4 or R9.5 of a MinION platform. The sequencing data were base-called in fast5 files and demultiplexed in fastq files. After removing the short and low-quality reads, the reads were aligned with the microorganism genome database. Species with identified reads higher than two for ONT data were reported.6/L). All three patients were positive by CSF India ink staining and CSF CrAg detection but negative by CSF culture  and increased white blood cell (WBC) counts  and NGS detected Cryptococcus in all three CSF samples. Nanopore sequencing produced 17,566 reads and 14,366 reads that were aligned to All three patients were treated with amphotericin B, flucytosine and fluconazole for 31–46 days, and the symptoms gradually improved. Thereafter, the patients were discharged and continued 12 weeks of outpatient follow-up. The symptoms of headache and fever gradually disappeared, and the haemogram returned to normal.CM is caused by inhalation of Cryptococcus from soil, pigeon droppings and rotting vegetation –3. CSF cC. neoformans, while NGS yielded 1,17,394 and 97,979 reads that were aligned to C. neoformans. The reads produced by nanopore sequencing were much longer than those produced by NGS. Thus, nanopore sequencing can sequence repetitive regions and structural variants, and provide comprehensive genomic information, such as the phenotype of antimicrobial resistance-encoding genes to help clinicians optimize the selection of antifungal agents for treatment (In this study, three patients were clinically confirmed to have CM by positive detection using CSF culture and India ink staining. Nanopore sequencing detected Cryptococcus in two CSF samples and NGS detected Cryptococcus in all three CSF samples. Nanopore sequencing produced 17,566 reads and 14,366 reads that were aligned to reatment , 19.This study was limited by the small sample size, and it is uncertain whether nanopore sequencing can increase the positive rate of pathogen detection. Therefore, further studies are needed.Nanopore sequencing with improvements in accuracy could change the diagnosis of infection in the future.


All six patients with suspected CSF leak showed paraspinal regions of significantly greater [18F]FDG uptake compared to the corresponding areas in controls (P < 0.05). Two patients treated with local injections  on the site of abnormal PET/MRI findings reported temporary but significant improvement in symptoms. Our results suggest [18F]FDG PET/MRI is sensitive to abnormalities potentially due to suspected CSF leak, which are not necessarily visible on conventional MRI alone or by the standard-of-care imaging methods.A combination of magnetic resonance imaging (MRI), computed tomography (CT), and radionuclide cisternography are typically used to locate a cerebrospinal fluid (CSF) leak. However, the site of leakage cannot be determined, making treatment more difficult. Therefore, more sensitive imaging tools are needed. A whole-body [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET)/MRI was conducted on six patients with suspected CSF leak and the resulting images were reviewed in comparison with those from six healthy controls. Paraspinal regions of focally increased uptake of [18F]FDG were quantified using maximum standardized uptake values (SUV The most characteristic symptom of a CSF leak is a persistent headache that worsens when upright and improves with recumbency (orthostatic headaches)2. When chronic, CSF leaks may present as “second half of the day headache” with patients consistently better in the morning after prolonged nocturnal recumbency followed by worsening symptoms as the day progresses with prolonged upright time3. Other symptoms include nausea, memory difficulties, cranial nerve dysfunction, neck and interscapular pain5. Hereditary disorders of connective tissue such as Marfan Syndrome, Ehlers-Danlos Syndrome, Neurofibromatosis type 1, and Polycystic kidney disease have been associated with spinal CSF leaks, suggesting these and other associated disorders of connective tissue may lead to abnormally vulnerable dura prone to CSF leak6. Among those who can identify a precipitating event, atraumatic activities such as stretching, or Valsalva maneuver are commonly involved9. Traumatic head/neck injury such as whiplash as well as iatrogenic dural tear during surgical interventions including lumbar puncture, durotomy, and skull base surgery are also important causes12. However, two thirds of patients with a spontaneous CSF leak can identify no precipitating events. The most common site of leak is along the spinal cord13. If the site of CSF leak is located, it is treated most commonly with an epidural injection of autologous blood  or an epidural fibrin patch, or surgery, depending on the case14.Cerebrospinal fluid (CSF) leak is a condition caused by drainage of CSF from the brain or the spinal cord through a hole in their surrounding structures, such as the skull and the dura. The prevalence rate of the CSF leak is estimated to be 1 in 50,000 individuals, but this relatively uncommon occurrence may be due to underdiagnosis15. Misdiagnosis is common, preventing proper treatment as the symptoms are often mistaken for those of other diseases, such as postural orthostatic tachycardia syndrome or new daily persistent headache16. The underlying sensitivity of magnetic resonance imaging (MRI) and computed tomography (CT) myelography remains unknown. While these two imaging modalities often are consistent in retrospective comparison17, this may reflect the common clinical scenario of using invasive CT myelography much more frequently in patients with positive MRI than in patients with negative MRI—attempting to localize the source of leak suggested by findings seen on the subset of positive MRI. In contrast, prospective studies of patients with orthostatic headaches suggest a potentially much lower sensitivity. In one study of 100 patients with orthostatic headaches suspected of having CSF leaks from a consortium of 11 hospitals in Japan, 70 had axial T2 spine MRI, finding 14 leaks, while 86 had MR myelograms, where only 3 leaks were noted18. For CT myelography, cases with slow or intermittent CSF leakage are very difficult to identify due to the insufficient contrast and resolution between CSF and background19. Previous studies estimate that 46–55% of these cases present no detectable signs of CSF leak on CT myelogram21. Thus, better non-invasive imaging techniques for detecting and localizing spinal CSF leaks are needed.Unfortunately, no one diagnostic test can consistently exclude the diagnosis of CSF leak or identify the site(s) of leakage22. We recently presented initial findings from our study23, and here we describe in detail our early experience of [18F]FDG PET/MRI of patients with chronic symptoms of suspected CSF leaks in comparison with healthy controls.In this report, we introduce the use of simultaneous [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET)/MRI to locate CSF leaks. We hypothesized that [18F]FDG PET, if coupled with the high spatial and contrast resolution of MRI, could accurately identify inflammation around the sites of CSF leak due to its pro-inflammatory natureIn all six patients, we observed sites of abnormally increased focal uptake of 18F]FDG in paraspinal structures at various levels of the spine  as summarized in Table 8FFDG in max in lesions from the patient group showed a higher mean value than the corresponding areas from the control group in all tissue types . On the other hand, abnormalities on MRI that might be related to CSF leak were identified in only half of the patients. In a limited number of patients followed for the treatment outcome, the patients whose blood-patch treatment sites covered the abnormalities identified by our method achieved better symptomatic relief than the patient whose treatment marginally included the identified abnormalities.In this report, we introduce a novel [18F]FDG PET/MRI approach for detecting the sites of CSF leak. While other nuclear medicine approaches such as radioisotope cisternography have been used to identify CSF leaks22. This CSF-induced irritation is reflected clinically in studies of patients with chronic CSF leaks following accidental dural puncture, who report much higher rates of spinal pain at the site of CSF leak than controls without dural puncture25. Lesions with active inflammatory responses can have increased metabolism and energy requirements, becoming more glucose-avid than normal tissues26. [18F]FDG PET is a highly sensitive imaging modality of glucose metabolism, and has demonstrated its effectiveness in detection of such hypermetabolic inflammation29. Our results show abnormally increased uptake of [18F]FDG in the paraspinal region of all patients, supporting the feasibility of our approach for the detection of CSF leak. More abnormalities were found with [18F]FDG PET than 3T MRI, which suggests [18F]FDG PET might be better suited than MRI for the detection of early inflammatory changes due to CSF leak. However, our approach may not be feasible for detecting other etiologies without extradural CSF inflammation, such as CSF-venous fistular where CSF escapes the thecal sac directly into adjacent epidural veins.The proposed approach for the improved detection of CSF leak is based on the observation that extradural CSF may irritate and inflame tissues outside of the nervous system30 has demonstrated that seromas typically present a thin rim of mildly increased [18F]FDG uptake surrounding a central area of no [18F]FDG uptake in contrast to ‘solid’ or ‘thick-walled’ [18F]FDG avidity of more aggressive lesions such as soft tissue sarcomas.Unlike the conventional imaging methods for CSF leak diagnosis, our [18F]FDG PET approach is to find the paraspinal tissues potentially affected by CSF leak rather than the tear or puncture through which the CSF leaks. Therefore, using [18F]FDG PET in conjunction with an imaging modality that enables the investigation of the anatomic cause of the leak can create a synergistic effect in accurately specifying the site(s) of CSF leak. MRI is more advantageous than CT for accompanying [18F]FDG PET in that it can provide detailed anatomic examination with no further deposition of ionizing radiation to sensitive areas. The simultaneous acquisition of PET and MRI employed in this study also offers improved image co-registration compared to the conventional PET/CT that sequentially acquires PET and CT, rendering it susceptible to mis-registration due to patient motion between the two scans. Our PET/MRI approach has the additional benefit of potentially discriminating a benign post-operative seroma from an extraspinal collection of CSF. On MRI, they are difficult to differentiate because they are likely to appear isointense to each other. However, on [18F]FDG PET, the benign seroma is expected to have minimal or no [18F]FDG uptake, while the extraspinal CSF collection is significantly [18F]FDG avid, such as the one seen in Patient 6 Fig. . Indeed,32. Therefore, careful consideration of other etiologies should be considered when abnormal uptake of [18F]FDG is observed in the paraspinal regions. Our early results suggest it could be effective as an additional tool for locating the sites of CSF leak and target sites for epidural patching. Optimizing MRI sequences that can better complement [18F]FDG PET is also necessary to pinpoint the site of CSF leaks. Additionally, post-treatment scans in these same patients will be useful in determining whether a decrease in symptoms correlates with a decrease in [18F]FDG tracer uptake.Our study has a few limitations. More study subjects are needed to determine whether this [18F]FDG PET/MRI approach is overall more sensitive and specific to CSF leaks than other imaging modalities. Uptake of [18F]FDG is relatively non-specific and can be seen in other paraspinal inflammatory processes such as myositis and in musculoskeletal degenerative changes such as Baastrup’s diseaseIncreased [18F]FDG uptake can be observed on PET in the paraspinal tissues of patients with suspected CSF leak. Our results suggest [18F]FDG PET/MRI is sensitive to abnormalities potentially due to suspected CSF leak, which are not necessarily visible on the standard-of-care diagnostic imaging modalities.All experimental protocols in our prospective observational research were approved by the institutional review board (IRB) of the Research Compliance Office, Stanford University, U.S.A. (protocol number IRB-24972). All methods were conducted in accordance with relevant guidelines and regulations.For the patient group, 4 male and 2 female patients suspected of suffering from CSF leaks were recruited . All patients presented with orthostatic headache and previously received standard-of-care imaging methods (including CT myelogram), which did not identify sources of CSF leak. The events identified by the patients as a precipitating event were epidural steroid injection (Patient 1 and 3), traumatic injury (Patient 4 and 5), lumbar puncture (Patient 6), and none (Patient 2). Six healthy controls  were also imaged with the same [18F]FDG PET/MRI protocol used for patients to compare the [18F]FDG uptake between the patient and control groups. Prior to imaging, all participants signed an informed consent form regarding study participation and publishing acquired data and images. All data were acquired in compliance with the Health Insurance Portability and Accountability Act. The clinical trial registration number of our study is NCT03195270 (registration date: 07/22/2017), and the name of the associated clinical trial registry is ClinicalTrials.gov.34.All subjects were requested to fast for 4 hours before our imaging study to avoid non-specific elevation of the blood glucose level. One hour after a single 10-mCi injection of [18F]FDG, subjects were admitted to a SIGNA PET/MRI scanner  for imaging. We performed a whole-body PET/MRI scan of subjects, which consisted of 8 to 10 consecutive imaging stations. In each station, 4–8-min simultaneous PET/MRI scan was performed, making the total scan time to be 1 to 1.5 h depending on the patient’s height. Acquired PET and MRI raw data were reconstructed using the algorithms equipped in the scannerIn each station, we conducted following two MRI sequences: 3D axial T1-weighted spoiled gradient-recalled echo sequence with two-point Dixon fat–water separation  and 2D axial T2-weighted fast-spin-echo sequence with two-point Dixon fat–water separation . For one or two imaging stations where we suspected CSF leaks might occur based on the patient’s history, we additionally ran the following two sequences for high-resolution imaging: 3D axial double-echo-in-steady-state (DESS) with water-only excitation  and 3D coronal motion-sensitized-driven-equilibrium (MSDE) fast-spin-echo sequence (CUBE) with triple-echo Dixon fat–water separation . For MRI signal acquisition, a 16-channel head-neck coil, an integrated spine coil, and two 32-channel anterior body array coils were employed.max) of [18F]FDG was measured. We used image analysis software  for measuring SUVmax. The lesions on the PET images were categorized into 5 types: paraspinal muscle, interspinous ligament, osseous tissue, neuroforamina, and fluid collection. The SUVmax measurements in each lesion type were compared with the SUVmax of corresponding areas in the healthy controls using a two-sided Mann–Whitney U-test. We employed this nonparametric test because we could not assume the normality in the measurements from patients and the sample size was small. P-value of 0.05 was adopted as a significance level.Two radiologists reviewed patient PET and MRI images concurrently to find abnormalities that are likely to be related with patients’ symptoms. Abnormal [18F]FDG hotspots were identified as potential lesions and the maximum standardized uptake value  following the treatments in comparison with previous treatments, if possible. We compared the sites of the blood patches with the sites of PET/MRI abnormalities from our study to assess the relevance of detected abnormalities to the symptom based on the patient’s post-treatment evaluation.After our imaging study, three patients received epidural blood-patch treatments based on the information from the standard-of-care diagnostic methods, without using our imaging findings. Patient 2 had targeted fibrin epidural patches while Patients 4 and 6 had targeted epidural blood patches. When a leak site has been identified, fibrin patches may resolve the symptoms of some patients with CSF leak refractory to epidural patching with blood


For conventional H+ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exiguobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H+ to the Schiff bases and then capture the other H+ from the medium, whereas the Lys donor first captures H+ from the medium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer reactions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H+ donor. In contrast, for Glu-type proteorhodopsin (PR) and ESR, the embedded residues functioned well as H+ donors. These differences were further examined by focusing on the activation volumes during the H+-transfer reactions. The results revealed essential differences between archaeal H+ pump (DR) and eubacterial H+ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H+-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.Many H They convert the energy of ATP hydrolysis or sunlight into electrochemical gradients of substrate ions across membranes. The resultant electrochemical gradients drive various metabolic processes and thus contribute to cellular homeostasis. Similar to other membrane proteins, the transmembrane regions of ion pumps consist of many hydrophobic residues. The resultant hydrophobic regions inside the proteins prevent unfavorable leakage of ions. However, these regions also make it difficult to accomplish ion transport tasks because once activated, ion pumps need to transport their substrate ions through hydrophobic regions. Thus, pumps seem to have some mechanism to overcome these difficulties. In this study, we analyzed such mechanisms of light-driven H+ pump rhodopsins belong to the microbial rhodopsin family, which is a huge family of photoactive membrane proteins widespread in unicellular microorganisms (via Schiff base (SB) linkage. Upon light absorption, the retinal undergoes isomerization from the all-trans to the 13-cis state, which in turn activates the protein by distorting the conformation. This activated state returns to the original state via various structural intermediates. During this cyclic reaction, called a photocycle, microbial rhodopsins exert various functions that are largely categorized into ion transporters and light sensors.The Hrganisms , 2, 3. T+ pump named bacteriorhodopsin (BR) from a highly halophilic archaeon , whose relatives were identified in marine bacteria inhabiting oceans worldwide   . As desc+ pump rhodopsins cause multiple H+-transfer reactions and finally accomplish H+ translocation across the cell membrane. + transfer steps for BR, PR, and ESR (lower panels) and their respective photocycling schemes (upper panels) . For BR, this primary reaction evokes subsequent H+ release to the extracellular (EC) medium from the “H+ release group (PRG)” . In PR and ESR, H+ release does not occur at this step because of the lack of PRG (B and C). Instead, they release H+ from the acceptor residue at the latter step (N decay). Thus, PRG is not essential for H+ pump function, and there are no important differences in the reactions at the EC side. In contrast, the reactions at the cytoplasmic (CP) side are distinct because of the difference in H+ “donor” residues (lower panels). Compared with the EC channels, their CP channels are highly hydrophobic. Thus, H+ pumps have special mechanisms to facilitate H+-transfer reactions, where the “donor” residues play central roles.Upon illumination, H panels) , 15, 16.+ pumps, the reprotonation of the SB occurs during M decay. This H+ is provided by the donor residue located approximately halfway within the CP channel. In BR and PR, Asp and Glu residues act as H+ donors   . Despite the different H+-transfer reactions, the carboxyl and Lys donors greatly accelerate M decay. Thus, these donors are commonly essential for efficient H+ pump function. The different H+-transfer reactions probably require different conformational changes. Thus, carboxylate-type and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer reactions.For all H panels) , 12, 13.r panel) , 14. Sim+ pump, we used deltarhodopsin (DR) instead of BR because DR can be functionally expressed in Escherichia coli despite its high similarity with BR    17). Fo. Fo+ pum+ from the medium passing through the hydrophobic channels. Thus, we replaced the donor residues and then examined their effects on M decay. Hereafter, we used the terms “donor-replacement mutants” and “donor-disabled mutants.” The former denotes the mutants whose donors were replaced by Lys or carboxyl residues, whereas the latter mutants have nondissociable residues at the donor positions.When the donor residue does not work, M decay becomes very slow because SB needs to capture HA and D) indicate the results for the wildtype proteins. In addition to the wildtype proteins, all prepared samples had λmax at approximately 520 to 550 nm, as shown in bottom panels (C and F). The middle panels (B and E) show the results for the Lys mutants (DR-D104K and PR-E108K). The replacements by Lys slowed down M decay to some extent. However, the decay rates were still significantly faster than those of the donor-disabled mutants. Thus, the embedded Lys residues appeared to function in DR and PR, even though the H+-transfer reactions should occur in the inverted order from the original reactions. For the donor-disabled mutants (bottom panels), other intermediates after M were not detected until the ends of the traces at 8 s because of the slow decay of M. However, for both the wildtype proteins and the donor-replacement mutants, the subsequent intermediates commonly appeared at longer wavelengths, reflecting the fast decay of M. For PR (D), the intermediate at 590 nm is assigned to N , N was short lived and did not significantly accumulate. Instead of N, the next intermediate O appears at 630 nm for DR as a result of reisomerization of the retinal to the all-trans state. O is the last intermediate, and its decay corresponds to the recovery to the original state. For PR, N transforms to PR', which is a precursor of the original state and contains all-trans retinal , M decay was completed within 10 ms. This fast decay was still observed after donor replacement by the Asp residue . Reflecting the fast M decay, the next intermediate appeared at 590 nm with almost the same kinetics as that of wildtype ESR. This intermediate was previously assigned to N for the wildtype protein because of the 13-cis configuration of retinal  slowed the M decay approximately 10-fold but was still significantly faster than that of the donor-disabled mutant . Thus, both carboxyl residues seemed to function as H+ donors, although they probably mediate different H+-transfer reactions from the wildtype ESR.The donor-replacement effects for ESR are summarized in Fig. 3 retinal . The repC and F and 3D). Thus, slow M decays significantly lower the photocycling rates, which should result in faint H+-pump activity under constant illumination. Conversely, we observed much faster photocycling rates for the donor-replacement mutants  (B and E and B and C). Thus, they should exhibit distinct H+-pump activities. E. coli suspensions. The outward H+ transport should decrease the pH values. These time traces are shown in Figure 4A, where the donor-disabled mutants exhibited only negligible pH changes (the bottom traces). On the other hand, distinct pH decreases were observed for not only the wildtype proteins but also the donor-replacement mutants. These pH changes disappeared after the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (broken lines), indicating that H+ was indeed pumped under illumination. To evaluate the relative pump activities, the initial slopes of the pH changes are plotted in Figure 4B after dividing by the relative amounts of the expressed proteins, which were evaluated by the flash-induced signals summarized in Figure 4C  exerted significant H+-pump activities similar to their wildtype proteins.For the donor-disabled mutants , M decay is the rate-limiting step of the photocycles  is replaced by a Gly residue, showed fast M decay because of the open structure of the CP channel even in the dark state  and 5 or 5.5 for ESR , the M decay rates were almost constant below pH 7, whereas above pH 8, the traces showed biexponential decays. The first decay rates were almost the same as those below pH 8, indicating that the donor (D104) still provided H+ to the SB at any pH. On the other hand, the second decay rates became slower as the pH increased. Essentially, the same pH dependences were also observed for BR , the M decay showed significant pH dependence at all measured pH values. This behavior is similar to that of the donor-disabled mutant , where SB directly captures H+ from the medium. Thus, the Lys residue does not function as a donor residue. for ESR  because dtype DR A, the M d for BR . The Asp N decay , 22, 23.DR-D104K B, the M t D104N; C, where E) exhibited an almost constant decay rate of M below pH 9. Thus, the Lys residue acts as an H+ donor at almost the same pH range as that of wildtype PR . Above the respective pH ranges , M decay commonly slowed down as the pH increased, as in the donor-disabled mutant . Thus, both donors are disabled at high pH, but the mechanism should be different between them. The M decay in wildtype PR did not show the biexponential decay that appeared in the wildtype DR . Thus, above pH 8, the Glu donor is probably deprotonated even in the dark states and thus does not contribute to M decay. On the other hand, the Lys donor in PR-E108K should be deprotonated at any pH in the unphotolyzed state. Thus, the dysfunction above pH 9 might reflect the fact that the Lys donor has a pKa of ∼9 for H+ uptake. Because of this pKa value, H+ uptake slows down above pH 9 and becomes the rate-limiting step for M decay.In contrast to DR, the embedded Lys residue surely functions in PR. The PR-E108K mutant E exhibitR <pH 8; D. Above A), the Lys donor ideally functions below pH 7, up to which point the M decay rate is almost constant. Similarly, both Asp and Glu residues (B and C) also confer constant M decay rates below pH 8. These three donors generally lost their ability at high pH, when the M decay slowed down as the pH increased, as in the ESR-K96Q (D). Similar to the case of PR, the mechanism should be different between Lys and carboxyl donors. For the Lys donor, its pKa for H+ uptake might be located near 7. As a result, M decay exhibited a pH dependence above pH 7. Carboxyl donors were probably deprotonated above pH 8 even in the dark states and thus did not contribute to M decay.Similar to PR, the embedded carboxyl residues also function in ESR. For wildtype ESR A, the Lyresidues , B and CESR-K96Q D. Simila+-transfer reactions mediated by the donor residue  exhibited a significantly large pressure dependence, similar to BR. Large pressure dependence was also observed for wildtype ESR (C). Conversely, PR and the mutant exhibited much smaller pressure dependence (B and D). The same tendency was also observed for the two ESR mutants (E and F). Thus, a smaller pressure dependence might be related to successful donor replacement.dtype DR A exhibittype ESR C. Converpendence , B and D mutants , E and Fk) is related to the associated activation volume (ΔV‡) by Equation k0. For the samples except for wildtype DR and ESR-K96E (A and E), decay of the next intermediate (N or N-like) was also fitted. Their traces (B–D and F) showed negative values before converging to zero. The negative values originated from large accumulations of N or N-like with smaller absorptions than those of the original dark states. Thus, their final increases reflected the decays of N or N-like. The smaller panels in ki) and the amplitude coefficients (Ai), for the phases of M decays and do not include the parameters for other phases (formation of M and decay of N or N-like). A complete set of the parameters is summarized in The decay rate constant , three exponents  were required to simulate M decay. Of them, the slowest component (k3) had a negligible amplitude (A3). Thus, we focused on the faster components associated with k1 and k2. At 1 bar, their corresponding time constants were approximately 1 and 6 ms, respectively. These components also appeared in k1, which roughly reflected the formation rate of MN equilibrium. The second decay rate became slower at higher pH and corresponded to k2, which roughly reflected the decay rate of MN equilibrium. Thus, biexponential decay was caused by the presence of a back reaction from N to M. As a result, both k1 and k2 involve complex contributions of rate constants for elementary processes, that is, M → N, N → M, and N → O. As shown later, similar biexponential decays were also observed for wildtype ESR and the K96E mutant. Conversely, for the other three samples ”  and “H+ uptake” (from medium to donor). Thus, km roughly reflects the rate for the completion of H+ transfers mediated by the donor residue. For DR, the logarithm of the km value is plotted in the small panel of Figure 7A together with the values for k1 to k3. From the slope, the activation volume for km was calculated and plotted in +-transfer reactions, similar to BR. The respective activation volumes for k1 to k3 were also calculated and summarized in For wildtype DR A, three C). The M decay phase at all pressures was fitted well with two decay rates (k1 and k2). Until 560 bar, the traces decayed beyond zero and then increased again, reflecting the accumulation of N and the subsequent decay. At these pressures, fourth-rate constants (k3) were needed to simulate the final increases in the traces. At other pressures, the traces were fitted well with three rate constants . The first-rate constant (k0) is associated with the formation of M. Thus, at all pressures, M decays were expressed by two rate constants, k1 and k2. For wildtype ESR, M was reported to achieve equilibrium with the subsequent N1  comparable to DR . The traces were only slightly slowed down under high pressure. As mentioned previously, the M decay phases were fitted well with a single decay rate (k1), whose value also slightly slowed down as the pressure increased . The lower pressure dependence provided a much smaller activation volume of 14.9 ml/mol compared with BR, DR, and ESR (k1 (M → N transition) reflects the rates of two H+ transfers by the donor, that is, “H+ donation” and the subsequent “H+ uptake” . The M decay of PR-E108K was also fitted well with a single decay rate, which involved two H+-transfer reactions, as for the wildtype PR, but their order was inverted because “H+ uptake” should occur first, followed by “H+ donation”. The faint pressure dependence resulted in a smaller activation volume of 9.5 ml/mol , the M decays were fitted well with two decay rates (k1 and k2), probably reflecting the formation of MN equilibrium and its subsequent decay. Similar to DR and ESR, we calculated km. The determined values showed a smaller pressure dependence compared with the wildtype ESR and thus led to a smaller activation volume of 31.0 ml/mol. For K96D (F), the M decays were well fitted with a single decay rate, whose pressure dependence was very small and led to a smaller activation volume of 22.5 ml/mol. It is not clear whether this M decay also involves H+ uptake, similar to wildtype PR. Even if this is not the case, the H+ capture process should also require a faint activation volume. H+ capture should occur during the decay of subsequent N- or N-like intermediate, which corresponds to the final increase in the traces. These traces did not show any pressure dependence.Significantly smaller pressure dependences were also observed for the two ESR mutants. For K96E E, the M For K96D F, the M + donor residue should confer a pH-independent “fast” M decay at least around physiological pH. The donor-replacement mutants of PR and ESR indeed exhibited the pH-independent M decays in respective pH ranges (B). Thus, Lys residue cannot act as H+ donor in DR. As mentioned previously, we initially expected that the donor replacements might not be allowed between carboxyl-type and Lys-type H+ pumps because of their optimized conformational changes for respective H+ donor residues. This view seems to be consistent with the results for DR-D104K. The Asp donor in DR should be first allowed access to the SB, but Lys donor needs to first access the CP medium to capture H+. Thus, conformational change of DR might disturb the H+-transfer reactions of Lys residue. However, this “mismatch” might be covered by the distorted protein structure. As shown in + inflow from the medium. The faint expression level of DR-D104K (C) might also reflect the steric effect. The CP channel of DR might be too tight to accommodate the Lys residue, so that its introduction might disturb structural formation in the cell membrane. Thus, “true” replaceability of the donor residue probably cannot be discussed for DR.The excellent HH ranges  and 6. TH ranges B. Thus, DR-D104K C might aB, PR-E108K exhibited significantly smaller H+-pump activity in spite of the fast M decay comparable to the wildtype PR .We believed that DR and PR commonly utilize the hydration for the Heactions A.Figure +-transfer reactions without significant hydration. Thus, ESR seems to share flexibility with PR. Indeed, previous X-ray structural analysis of ESR revealed some extent mobility of the donor residue, where the Lys side chains took different conformations in the antiparallel ESR dimers with different electron densities , we assumed that the swing-like motion of the donor is a key mechanism not only for the wildtype ESR but also for the donor-replacement mutants. This common mechanism might be the reason that successful donor replacements were achieved.Is high flexibility also true for the wildtype ESR and the mutants? Smaller activation volumes for two mutants (ESR-K96E and ESR-K96D) suggest a common mechanism with PR, that is, the embedded Glu and Asp donors seem to exert a swing-like motion and facilitate the Hensities . Based on for PR B, we ass+ donor, similar to Glu and original Lys residues. However, the Asp side chain is shorter than the other side chains. Thus, for Asp residue, the swing-like motion might not be enough to facilitate the H+ relay reactions. This disadvantage might be overcome by the displacements of the water molecules inside the protein. The Lys-to-Asp replacement is likely to create the cavity, which is probably filled by the water molecules in the dark state. In the photolyzed state, these water molecules might move together with the Asp side chain to assist the H+-transfer reactions. This mechanism does not involve further hydration and the resulting volume increase.In ESR, the embedded Asp residue can act as an excellent H+ pumps, PR and ESR appeared to cause smaller hydration of the CP channel. Especially, PR and its E108K mutant exhibited much smaller activation volumes, indicating that only slight hydration occurred during the M decay. Different amounts of hydration should originate from different conformational changes. In the photolyzed BR, the CP portion of helix F causes outward tilting and then induces the hydration of the CP channel. The initial key events for this conformational change are considered to be the displacement of the 13-methyl group of the retinal and the twist of the Lys216 side chain, which connects the retinal with helix G via SB linkage . Ev+ pumping mechanism have been vigorously analyzed. Concerning the donor residue, its H+-transfer reactions were revealed to require a large amount of hydration of the CP channel  and inserted into the pKA001 vector under the lacUV5 promoter with NdeI and HindIII sites , UT5600, and BL21 were used for the wildtype and mutant DR, PR, and ESR, respectively. The respective procedures were essentially the same as those previously described -arabinose for PR. After 3 to 4 h of induction, the cells were harvested and broken with a French press. The collected cell membrane fractions were solubilized with 1.5% n-dodecyl β-D-maltopyranoside, and the solubilized proteins were purified using nickel–nitrilotriacetic acid agarose. The concentrations of the proteins were determined from the absorbances at λmax values of approximately 520 to 540 nm by assuming extinction coefficients of 63,000 M−1 cm−1 for DR and 50,000 M−1 cm−1 for PR and ESR. The purified samples were replaced with an appropriate buffer solution by passage over Sephadex G-25 in a PD-10 column (Amersham Bioscience).For expression and purification, escribed , 35, 36.Lipid reconstitution was performed as previously described . For rec2SO4 or NaOH.The flash photolysis apparatus equipped with a neodymium-doped yttrium aluminium garnet laser  was described previously , 39. TheFlash photolysis under various pressures was measured by replacing the sample holder with a pressure cell, whose details were described previously . The cylk) of the intermediate has the following relationship with the associated activation volume (ΔV‡):P, R, and T denote the hydrostatic pressure, gas constant, and absolute temperature, respectively. To determine the rate constant, we fitted the time traces of absorption change (ΔA(t)) with the following multiexponential functions:Ai and ki denote the amplitude coefficient and decay rate constant, respectively. The M decays of DR, ESR, and the ESR-K96E were expressed by two rate constants, k1 and k2. For these H+ pumps, the following “mean rate constant (km)” was introduced:τm, τ1, and τ2 are the time constants and reciprocals of km, k1, and k2, respectively.The decay rate constant . The light intensity was adjusted to approximately 24 mW/cm2 at 530 nm using an optical power meter . All measurements were performed at room temperature (approximately 25 °C).The Hreported . Briefly+-pump activities were evaluated by the initial slopes of pH decreases upon illumination. However, these slopes depend on the expression levels of the H+ pump rhodopsins. The expression levels were estimated by the magnitudes of the flash-induced absorbance changes. The procedures were previously described (+-pump activities, the cells were collected by centrifugation and then suspended in 2 ml of 50 mM Mes, pH 6, and 200 mM NaCl. After disrupting the cells by sonication, the lysates were used for the measurements of flash-induced absorbance changes. The measurement wavelengths were set to the λmax values of the absorption spectra in the n-dodecyl β-D-maltopyranoside–solubilized state. The negative deflected signals reached maximum values within 0.1 to 10 ms. These values were employed as the relative expression amounts of the H+ pumps. To calculate the relative H+-pump activities, the initial slopes of pH decreases were divided by the maximum values of the flash-induced absorbance changes.The Hescribed . After tAll the data supporting the findings of this study are available within the article and supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Imbalance of Aβ and tau protein production and clearance are the key factors among many causes of Alzheimer's disease that leading to neurons degeneration and cognitive disorders. As a novel approach, glymphatic system quickly clear metabolic waste  from cerebral environment, and dysfunction of glymphatic system may relate to occurrence of Alzheimer's disease. Microinfarct is a common histopathologic situation occurring in aging brain and leads to dramatic increase the generation of metabolic by-product after neuronal injury, hindering the operation of glymphatic system and suppress cerebral spinal fluid (CSF) and cerebral interstitial fluid  exchange. Microinfarcts destruct the integrity of microvascular and microstructural tissue, result in Aβ deposition and tau phosphorylation that form neurofibrillary tangles and associated with the cause of Alzheimer's disease. Currently, it has been found that glymphatic system is involved in the pathological process of Alzheimer's disease. Improving the function of glymphatic system after cerebral microinfarcts could be developed as a new approach for Alzheimer's disease prevention and treatment. In this review, we will provide in-depth discussion on functional changes of glymphatic system after cerebral microinfarcts, further reveal pathogenesis of Alzheimer's disease and provide a potentially more effective method for treatment of Alzheimer's disease. Cerebral microinfarct is a common pathological event in patients with dementia and other cerebrovascular diseases Glymphatic perfusion of the central nervous system (CNS) serves multiple purposes in neurophysiology. It is important for delivery of nutrients, specifically glucose Aqp4 knockout mice compared with wild-type mice Alzheimer's disease (AD) is associated with the accumulation of toxic aggregates of Aβ and hyperphosphorylated tau in the brain. The concentration-dependent aggregation of Aβ peptides is determined by the balance between production and clearance mechanisms. Aβ peptides are generated from the amyloid precursor protein. Aβ can be cleared from the brain by several mechanisms, including intracellular and extracellular degradation, accumulation in plaques, transcytosis across the blood-brain barrier, and bulk clearance into the CSF. Unlike Aβ, tau is an intracellular protein, hyperphosphorylated tau is thought to be generated in the late stages of neurodegeneration in AD. The spread of tau follows the pattern of neuronal connectivity; therefore, clearance of tau from the brain is much slower than that of Aβ. AQP4-dependent glymphatic pathway for driving the removal of soluble Aβ from the interstitium is an important clearance system. In mice, Aβ is cleared along perivascular, and Aβ clearance was reduced by 55-65% in As a key component of the central lymphatic system, glymphatic system plays an important role in Alzheimer's disease (AD), brain injury (TBI), microinfarcts and other cognitive disorders. Here, we will discuss whether the pathogenesis between neurodegenerative diseases (such as Alzheimer's disease) and cerebral microinfarcts are caused by changes in glymphatic function after microinfarcts. This complex mechanistically glymphatic system could provide new therapeutic targets for neurological disorders associated with immune malfunction and protein aggregation, leading to new therapies.Microinfarcts are a common feature existing among elderly patients, particularly in those suffering mild cognitive dysfunction, vascular or Alzheimer's dementia and are thought to contribute to cognitive decline in elderly patients In addition to causing local tissue damage, microinfarcts can also lead to neurodegenerative changes in distal brain regions Data compiled from studies suggest that microinfarcts occur throughout brain, including cortex and subcortical gray and white matter. All cortical and subcortical areas are found to have microinfarcts by comparing incidence of lesions in cortical and subcortical areas. The microinfarcts are then assessed and provide a more complete description of microinfarcts in gray and white matter. The result of two studies specified that no difference was found in appearance of microinfarcts between cortical and subcortical regions, while another study showed that microinfarcts were more common in subcortical regions. However, whether microinfarcts occur preferentially in cortex or subcortical areas was not discussed in most of the studies. Regarding to specific brain areas, cerebral microinfarcts are most common in parietal and occipital areas. Others are only in cortical gray matter. White et al. Thus, these results suggest that microinfarcts can occur in all brain regions. However, the occurrence of lesions in different brain regions was not compared specifically, but some studies found microinfarcts were more abundant in cerebral cortex.Various underlying causes led to microinfarcts and were able to co-exist in a patient. Three of the main causes are cerebrovascular disease , microemboli and hypoperfusion. Researches on neuropathology discovered that cerebral microinfarcts are often accompanied by occurrence of severe cerebral amyloid vascular disease Neuropathologic studies further explored the relationship between cerebral microinfarcts and cerebral amyloid angiopathy. A study with brain examination of about 1000 community residents, it was found that cortical microinfarcts were accompanied by cerebral amyloid angiopathy Besides cerebral amyloidosis, cerebral microinfarcts have been found accompany with other small vascular diseases by MRI. Cerebral microinfarcts co-occur simultaneously with lacunar infarcts and were tied in with higher white matter volume and higher cerebral atrophy A 3T MRI study indicated that cerebral microinfarcts were not associated with diabetes or hypertension, but were significantly associated with hyperlipidaemia Neuropathologic studies have verified the association between cerebral microinfarcts and dementia and cognitive impairment, but the relationship between cerebral microinfarcts and other concurrent pathologies are complex and might require further investigation Altogether, these in-vivo MRI studies support neuropathological findings that cerebral microinfarcts are related to poor cognition and are not affected by other age-associated pathologies. However, it is unclear whether these associations are causal or not. Because cerebral microinfarcts are widely distributed throughout the brain, these lesions might cause enough disruption leading to functional impairment, which has been supported by animal studies Cerebral arterial circulation consists of anterior cerebral circulation supplied by internal carotid artery, vertebral artery and posterior cerebral circulation. The anterior circulation consists of the middle cerebral artery and the anterior artery, which are connected to posterior circulation through basilar artery, and the posterior cerebral artery by anterior and posterior communication arteries of the Circle of Willis Blood enters post-capillary venules from the cerebral capillaries, where the basement membranes of endothelial cell and astrocytes expands and provide a space around vessels for CSF drainage. Generally, blood from interior of the brain flows into larger central/deep veins and out of the cerebral cortex and subcortical white matter through cortical veins that extend to brain surface Recent studies have shown continuous exchange of cerebrospinal fluid and interstitial fluid (ISF). CSF enters the Virchow-Robin spaces from subarachnoid space through a combination of arterial pulse respiration and CSF pressure gradient. The subsequent transport of CSF into dense and complex brain parenchyma is driven by AQP4 water channels. The AQP4 channel is expressed in a highly polarized manner in astrocytic end-feet, which opens up brain vasculature Two-photon microscopy was applied to characterize the dynamics of the glymphatic system in mice by injecting fluorescent tracer labeled CSF into a large cisterna Therefore, we postulate that central nervous system (CNS) is the only organ system that lacks lymphatic vessels to assist in the removal of interstitial waste. Recent studies have identified the glymphatic system, a glia-dependent perivascular network that assists lymphatic function in the brain In an experiment with Oa-45 tracer injected into the lateral ventricle observed tracer flow from ISF to CSF through glymphatic system, and further transferred to neck lymph nodes and into blood, indicating that extracellular solute of brain cells can be cleared out of the brain through glymphatic system. Amyloid β (Aβ) injected into the brain of mice can be rapidly cleared by glymphatic system Entry of CSF through the perivascular space is essential for glymphatic ISF-CSF exchange and clearance function. Glymphatic transportation of CSF along the periarterial space, then the convective flow along the brain parenchyma, and outlet of interstitial fluid (ISF) along the perivenous space to neck lymphatic system is an energy-demanding process driven by a variety of mechanisms. The choroid plexus constantly produces CSF, creating a pressure that indicates the direction of fluid flow through ventricular system to subarachnoid space. What drives CSF into the pial along the perivascular space? Particular to arteries, pulsation produced by the smooth muscle cells generate impulses along the entire length of the pial meninges arteries and penetrate from the cortical surface into the perforating arteries of brain. Dobutamine, an adrenergic agonist, significantly increased pulsatile effect of mice and caused a flood of cerebrospinal fluid into parenchyma. Internal carotid artery ligation inhibited the pulsation in the opposite effect. In addition, reduction of pulse waves reduced the exchange of CSF-ISF AQP4, which is highly polarized and expressed on the end-feet of astrocytes, acts as a barrier between glymphatic system and brain parenchyma and plays a crucial role in regulating fluid flow between astrocytes, CSF and blood vessels In glymphatic system, ISF and CSF converge in brain parenchyma. During sleep, the intercellular space of brain cells increased by more than 60%, and the convection exchange between ISF and CSF also increased significantly, which accelerated the clearance of Aβ in CSF and made the concentration of Aβ in CSF during sleep lower than that during awake Body position can affect exchange efficiency of CSF and ISF through the change of hemodynamics, thus affecting glymphatic system. It was found that the material exchange efficiency of CSF and ISF was the highest in the right lying position and the lowest in the prone position. The reason may be that the heart position is higher in right lying position, which can promote blood suction and venous return, increase cardiac output, improve cerebral artery pulsation and thus improve the material exchange efficiency of CSF and ISF; while in prone position, the head is relatively higher, CSF outflow along neck vessels increases, which can reduce CSF in the brain, resulting in decreasing of the material exchange efficiency of CSF and ISF In the glymphatic system, PVS surrounds cerebral artery, CSF or CSF-ISF mixture flows in PVS, and cerebral artery pulsation can promote rapid movement of solute in PVS in the form of mixing and diffusion, thus completing the material exchange between CSF and ISF Microinfarcts destroy the integrity of microvessels or microstructures, reduce exchange of substances between CSF and ISF, promote Aβ and high phosphorylation of tau proteins deposits to form neurofibrillary tangles, and increase the risk of dementia. Dysfunction of glymphatic system leads to inflammatory responses, neurodegeneration, and cognitive impairment The core of a cerebral microinfarct is filled with microglia and surrounded by reactive astrocytes. AQP4 highly polarized and expressed on astrocytes terminal feet, is the barrier between glymphatic system and brain parenchyma and plays an important role in regulating astrocytes and the flow of fluid between CSF and blood vessels It has been reported that deletion of AQP4 gene leads to damaged posttraumatic glial scar formation and affects migration of astrocytes in primary cell culture Microinfarcts are common features in elderly patients, particularly those suffering mild cognitive decline or Alzheimer's disease, and some are thought to play a crucial role in age-related cognitive decline. However, the time course of individual development remains obscure because the microscopic infarcts are typically first identified only upon post-mortem examination. Microinfarcts described in the human clinical are incomplete and diffuse, lesion cores were associated with CD68-positive microglia and GFAP+ astrocyte processes, were typically microscopic (<1mm). GFAP-immunoreactivity and AQP4 polarization within perivascular end-feet, suggesting that water homeostasis and edema clearance may be compromised for a long period of time within a wide region surrounding these diffuse ischemic lesions. AQP4, which is localized to astrocytic end-feet, plays a role in CSF-ISF exchange and interstitial solute clearance through perivascular drainage pathways. The vascular polarization of astrocytic AQP4 is partly lost in reactive astrocytes in diffuse microinfarction brains. The finding that β-amyloid accumulation after microinfarcts was associated with loss of perivascular AQP4 polarization, and that presence of cortical parenchymal AQP4 is associated with CSF-ISF exchange, the microinfarcts related decline in glymphatic function might be in part attributable to dysregulation of astroglial water transport.There is increasing evidence that reduction of microinfarcts in glymphatic clearance contribute substantially to the accumulation of protein aggregates. Glymphatic system, as a metabolic waste discharge pathway and material delivery system of brain, plays an important role in CSF circulation by promoting the production of CSF and keeping the perivascular space unobstructed which leads to brain homeostasis. Polarizing distribution of AQP4 increase cell gap of brain, improve function of glymphatic system, which is expected to be the target of promoting the clearance of Aβ and tau protein. After microinfarcts, astrocyte activation, AQP4 polarity distribution disappeared, impaired glymphatic pathway function causes a wide range of neural and network damage, the disruption of solute transport surrounding these lesions may similarly drive outsized changes in neuronal network function because of the close association of deep lesions with the underlying white matter. The failure of glymphatic system clearance in microinfarcts might lead to accumulation of β-amyloid and hyperphosphorylated tau proteins and render the brain more vulnerable to developing neurodegenerative pathology or escalate the progression of cognitive dysfunction.More recent studies have revealed that β-amyloid and fibrillary tangles of tau in Alzheimer's disease are present in the interstitial fluid and CSF. The production and turnover of β-amyloid is strikingly rapid in humans. β-amyloid is not only produced by neurons. in fact, all cells produce β-amyloid. Bulk clearance by the glymphatic system might provide necessary and sufficient removal of extracellular β-amyloid until the end-of-life which time failure in adequate CSF bulk flow leads to accumulation of β-amyloid. This suggests that low activity of the glymphatic system could be a major risk factor for development of neurodegenerative diseases. Cerebral microinfarcts activated astrocytes resulting in loss of AQP4 which might reduce glymphatic CSF influx and the stagnation of CSF influx might accelerate β-amyloid accumulation. In turn, a spiral of protein accumulation, deformation of glymphatic routes and further reduction in protein clearance and pathology.Glymphatic dysfunction characterized by a failure of interstitial solute clearance is a central feature of aging brain, as well as CNS diseases including Alzheimer's disease, TBI and microinfarcts. While much is known about the regulation of glymphatic pathway, including the roles of cerebral arterial pulsatility, state of consciousness, and head position. There are no glymphatic therapies to intervene in these disease processes at present. The primary assignment of future studies will be identification of a target for regulation CSF-ISF exchange within the glymphatic pathway, to promote improved solute clearance in these diseases.


Neurofilament light chain (NFL) measurement has been gaining strong support as a clinically useful neuronal injury biomarker for various neurodegenerative conditions. However, in Alzheimer’s disease (AD), its reflection on regional neuronal injury in the context of amyloid pathology remains unclear. This study included 83 cognitively normal (CN), 160 mild cognitive impairment (MCI), and 73 AD subjects who were further classified based on amyloid-beta (Aβ) status as positive or negative (Aβ+ vs Aβ−). In addition, 13 rats (5 wild type and 8 McGill-R-Thy1-APP transgenic (Tg)) were examined. In the clinical study, reduced precuneus/posterior cingulate cortex and hippocampal grey matter density were significantly associated with increased NFL concentrations in cerebrospinal fluid (CSF) or plasma in MCI Aβ+ and AD Aβ+. Moreover, AD Aβ+ showed a significant association between the reduced grey matter density in the AD-vulnerable regions and increased NFL concentrations in CSF or plasma. Congruently, Tg rats recapitulated and validated the association between CSF NFL and grey matter density in the parietotemporal cortex, entorhinal cortex, and hippocampus in the presence of amyloid pathology. In conclusion, reduced grey matter density and elevated NFL concentrations in CSF and plasma are associated in AD-vulnerable regions in the presence of amyloid positivity in the AD clinical spectrum and amyloid Tg rat model. These findings further support the NFL as a neuronal injury biomarker in the research framework of AD biomarker classification and for the evaluation of therapeutic efficacy in clinical trials. N  is not specific to AD but provides staging of AD pathophysiological processes and can be measured using magnetic resonance imaging (MRI), [18F]FDG-PET, and CSF total tau (T-tau) FDG-PET or MRI scans [To maximize the utility of a novel biomarker and its proper use in AD research and therapeutic trials, it is imperative to compare it with other well-established biomarkers. In the case of NFL, this would facilitate its incorporation among the existing biomarkers . CurrentRI scans , 22. ThiRI scans . UnderstIn this study, we elucidate the contributing role of Aβ pathology on neuronal injury reflected by NFL concentration and grey matter density maps. In addition, we illustrate voxel-based correlates between NFL and grey matter density maps in humans and McGill-Thy1-APP transgenic (Tg) rat model . This Tgadni.loni.usc.edu). The ADNI was launched in 2003 by the National Institute on Aging (NIA), the National Institute of Biomedical Imaging and Bioengineering (NIBIB), the Food and Drug Administration (FDA), private pharmaceutical companies and non-profit organizations, as a $60 million, 5-year public–private partnership. ADNI is the result of efforts of many co-investigators from a broad range of academic institutions and private corporations, and subjects have been recruited from over 50 sites across the USA and Canada. For up-to-date information, see www.adni-info.org.Data used in the preparation of this article were obtained from the ADNI  classification are mini-mental state examination (MMSE) scores between 24 and 30 (inclusive) without cognitive complaints or observable anomalies. The criteria for the diagnosis of mild cognitive impairment (MCI) included the presence of memory complaints, MMSE scores between 24 and 30 (inclusive), objective memory loss, Clinical Dementia rating (CDR) of 0.5, preserved activities of daily living, and absence of dementia. Mild AD dementia subjects had MMSE scores between 20 and 26 (inclusive), CDR of 0.5 or 1.0, and fulfilled the NINCDS/ADRDA criteria for probable AD.All procedures described here were performed in compliance with ethics protocols approved by McGill Animal Care Ethics Committee and following the Canadian Council on Animal Care guidelines. All rats were housed at the Douglas Mental Health University Institute animal facility on a 12/12 h light/darkness cycle with ad libidum access to food and water. A starting sample of 26 animals was included in this study based on the effect size estimated from the previous study [www.adni-info.org). For our study, we included 83 CN, 160 MCI, and 73 AD individuals who have both CSF and plasma NFL, CSF Aβ42, CSF phosphorylated tau (p-tau), and structural MRI data available. For plasma NFL analysis, we identified two CN, eight MCI, and four AD subjects as outliers (two standard deviations away from the mean) who were excluded in the analysis.ADNI inclusion and exclusion criteria can be found at . Last, all animals that were identified as an outlier (two standard deviations away from the mean) were excluded (two WT).http://adni.loni.usc.edu/methods). All T1-weighted MRIs were processed using an in-house processing pipeline and underwent non-uniformity correction [www.bic.mni.mcgill.ca/ServicesSoftware) generated Voxel-based morphometry (VBM) images representing grey matter density maps. In brief, a log Jacobian determinant was derived based on the nonlinear vector field from the previously mentioned pipeline. Then, it was transformed into a scalar, modulated with grey matter probability mask, and smoothed with 8 mm full-width at half-maximum (FWHM). Last, three ROIs from the MNI template atlas were used to replicate the previous findings in the literature: (1) temporal composite , (2) Entorhinal cortices, and (3) hippocampi [Details on the image acquisition parameters can be found here . A root-mean-square image of eight phases acquisition was performed. Each angle acquisition was acquired in a field-of-view of 36 × 36 × 36 mm with a matrix of 180× 180× 180× of TE/TR of 2.5/5.0 ms with a flip angle of 30° and NEX of 2. The final image was an average of the 16 acquisitions with a final 250 µm isotropic resolution and a total acquisition time of 46 min scan.www.bic.mni.mcgill.ca/ServicesSoftware). All individual MRI images were aligned with rigid body transformation, affine, and nonlinear transformation, and matched to generate an average population-based template based on Pydpiper [All images were processed using MINC toolkits (Pydpiper .Nonlinear vector field was used to create a log Jacobian determinant for deformation-based morphometry (DBM) representing local volume difference in each subject . Considewww.adni-info.org). CSF Aβ42 and CSF p-tau concentrations were measured using the Elecsys on a cobas e 601 analyzer as described previously [42 ratio cutoff threshold (CSF p-Tau/Aβ42 > 0.025) [CSF collection, processing, and storage procedures have been described previously . In brief, plasma NFL concentration was measured using an in-house single-molecule array (Simo) method, as described previously in detail [Plasma NFL collection, processing, and storage procedures have been described previously  were collected via direct puncture through the cisterna magna at 10–18 months of age.Rat CSF NFL concentration was determined using the in-house Simoa NFL assay which has been described in detail previously , with so42 and Aβ40 were measured using Quanterix kits according to kit instructions  with a CSF dilution of 100× using the provided assay diluents. For Aβ42 CSF measurements, the average repeatability coefficient of variation of a sample with a mean concentration of 1650 pg/mL was 3.2% and intermediate precision was 9.2%, and for a sample with a mean concentration of 641 pg/mL, repeatability was 10.9% and intermediate precision was 16.6%. For Aβ40 CSF measurements, repeatability for a sample with a concentration 5160 pg/mL was 17.2%, and intermediate precision 17.2%, and for a sample with a concentration of 3975 pg/mL repeatability and intermediate precision was 7.3% and 11.8%, respectively. For both assays, a number of samples were below the assigned assay limit of quantification and assigned a concentration of half of the LLoQ.Rat CSF concentrations of Aβt test. We log transformed NFL in CSF and plasma, as well as CSF Aβ42 and p-tau.Potential difference in the number of male and female in each group was tested based on a chi-square test for both humans and animals as well as APOEε4 status in humans. We also tested whether there were differences in age, education, and MMSE scores for humans based on a two-sided ANOVA. We tested if WT and Tg had any difference in age using an unpaired two-sided 42/40 and CSF NFL log: (1) CSF Aβ42/40 ~ Age + ; (2) CSF NFL (log) ~ Age (WT or Tg) + ; (3) CSF NFL (log) ~ Age + Genotype + Age × Genotype + . The association between CSF NFL and CSF Aβ42/40 was investigated based on a linear regression model. Weight was retained as a variable only in the model showing the association between CSF NFL and CSF Aβ42/40 as it was shown to have a significant effect.In animals, linear mixed effect models with a random intercept were performed to measure longitudinal changes in CSF Aβ42:p-tau interaction term was tested to exclude the effect of p-tau and Aβ42 interaction when the term was significant.In animals and humans, voxel-based linear regression models were performed to detect the group contrast in DBM and VBM, respectively. Furthermore, a voxel-based linear regression was performed between DBM and CSF NFL log at 15 months for animals, and VBM and CSF or plasma NFL levels for humans: animal model: CSF NFL log ~ DBM; human model: NFL (CSF or plasma) ~ VBM + covariates in each group as well as combining all groups with adjusting for the diagnosis. Similarly, we used those same models as the voxel-based models to replicate the previous findings using the Temp.Comp, entorhinal, and hippocampus ROIs with FDR correction for animals and humans , 17, 20.p = 0.05 and cluster threshold at p = 0.05 for rats and p = 0.005 for human [All voxel-based analyses were conducted using VoxelStats and corrected for multiple comparison using random field theory with peak threshold at or human . All theTable For the animal data, there was no difference in age, sex, or weight Table .42/40 from 10 months to 18 months  = 0.0045, t(63) = −2.09, p = 0.041)  = 4.06, p = 0.0004) and Tg  = 5.63, p < 0.0001)    4.38, p < 0.0001) while Tg had a significantly greater CSF NFL concentration compared to WT  = 2.14, p = 0.035)  = −2.40, p = 0.02)  Fig. . On the 01) Fig. . When we35) Fig. . However02) Fig. .Fig. 1Nat(11) = 3.2, p = 0.008), bilateral dorsal hippocampi (t(11) = 2.3, p = 0.042), and striatum in Tg compared to WT with the greatest contrast seen in thalamus (t(11) = 5, p = 0.0004)  = 13, p = 0.0001) and hippocampus (t(6) = 8, p = 0.0002)  = −3.18, p = 0.037), entorhinal cortex  = −2.99, p = 0.037), and hippocampus VBM  = −2.47, p = 0.048) only in Tg while WT showed no association. , medial frontal cortex, and lateral temporal cortex Fig. . The gret(99) = −3.3, p = 0.0034) in MCI Aβ+  = −2.38, p = 0.018), entorhinal cortex VBM  = −2.51, p = 0.015), and both CSF and Plasma NFL were associated with hippocampus VBM  = −4.59, p < 0.0001; β = −0.70, s.e = 0.24, t(99) = −3.03, p < 0.013, respectively) in MCI Aβ+  = −2.69, p = 0.016) and hippocampus VBM  = –3.99, p < 0.0001) while plasma NFL and VBM in the Temp.Comp and hippocampus ROIs showed a small trend (t(61) = −1.88, t(61) = −1.81, respectively, p = 0.15).In AD Aβ+, both CSF and plasma NFL levels were significantly associated with VBM results in dorsal lateral frontal cortex, lateral temporal cortex, precuneus/PCC, medial frontal cortex, angular gyrus, hippocampus, and cerebellar grey in the voxel-based analyses Fig.  and 11. Although CN Aβ− and Aβ+ and MCI Aβ− groups showed a significant association between NFL and VBM, the RFT-survived clusters were small. CN Aβ− showed a negative association between CSF NFL and lateral front-orbital cortex while CN Aβ+ showed in the cingulate cortex  that also are invariably present in clinically manifested AD, it is difficult to fully dissect the degree of contribution from Aβ alone on NFL concentration , 18, 37.42/40 significantly decreased while CSF NFL concentration was significantly increased in Tg animals, showing the inverse relationship between the two biomarkers, thereby replicating the human finding [In addition, our novel findings from the first longitudinal animal CSF analyses revealed that the CSF NFL concentration significantly increased during the healthy aging process as indicated by the results in WT. Also, the CSF Aβ finding . Conside finding , 26, 39. finding . Therefo finding . The dis finding , 40. Non42:p-Tau interaction that could potentially confound the association between NFL levels and grey matter density maps, and yet, we found correlations only in the Aβ positive groups and in the AD-vulnerable regions [Neuronal injury or neurodegeneration is not an AD specific process. Besides AD, increased levels of NFL are also reported in other neurodegenerative conditions such as frontotemporal dementia, progressive supranuclear palsy, and Creutzfeldt-Jakob disease , 41, 42. regions . The reaThere are some important limitations to this study. Due to the modest effect size in the association between NFL concentration and grey matter density maps, the available number of individuals who have both CSF and plasma NFL concentration and MRI data in the ADNI database is overall low, which limited the statistical power of our study. Perhaps this weakness could explain why we observed a minimal association in CN Aβ+. Future studies with a greater number of participants and longitudinal data would be of interest. Another possible reason for the minimal association in CN Aβ+ may be because some CN elderly individuals with high Aβ pathology might actually have minimal levels of toxic soluble Aβ oligomers . Indeed,In summary, our evidence supports that elevated NFL levels in AD results from Aβ-induced neuronal injury in AD-vulnerable regions. NFL is a sensitive biomarker capable of detecting the modest levels of neuronal injury linked to the healthy aging process or to pure Aβ pathology. As such, our study supports the hypothesis that NFL is a novel biomarker of neuronal injury and neurodegeneration that may be utilized within the A/T/N classification scheme and for the evaluation of therapeutic efficacy in clinical trials.Supplementary TablesSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7Supplementary Figure 8Supplementary Figure 9Supplementary Figure 10Supplementary Figure 11Supplementary Figure 12Supplementary Figure 13Supplementary Figure 14Supplementary Figure 15Supplementary Figure 16Supplementary Figure 17Supplementary Figure Legends


From the initial stages of the pandemic in early 2020, COVID-19-related olfactory and gustatory dysfunctions have been widely reported and are emerging as one of the most frequent long-term sequelae of SARS-CoV-2 infection. However, data regarding the long-term recovery of the sense of smell and taste are lacking. This study aimed to characterize the evolution up to one year after the diagnosis of self-reported olfactory and gustatory dysfunctions in COVID-19 cases.Based on the data of the active surveillance platform of the Apulia region, Italy, we selected the residents of Foggia district who were confirmed positive for SARS-CoV-2 from March 1st to June 16th, 2020, and home-quarantined with paucisymptomatic-to-mild clinical presentation. Self-reported olfactory and gustatory dysfunctions were recorded at baseline through a survey of dichotomous questions. The evolution of these symptoms at approximately one year was prospectively assessed via telephone by the validated sino-nasal outcome test 22 .Among the 1,175 COVID-19 cases notified in the Foggia district during the first epidemic wave, 488 had paucisymptomatic-to-mild clinical presentation. Of these, 41.2%  reported at least one sensory dysfunction. A total of 178 to 201 (88.5%) patients agreed to participate in the follow-up survey. According to the SNOT-22 results, the persistence of a sensory dysfunction was observed in the 29.8%  of them. Particularly, loss of smell persisted in 25.8% , loss of taste in 21.3% , loss of both in 17.4%  of participants in the follow-up. The rates of full recovery increased over time: from 59% at 30 days to 71.9% at 90 days for the sense of smell; from 61.3% at 30 days to 74.7% at 90 days for the sense of taste.The persistence of COVID-19-related olfactory and gustatory dysfunctions up to 12 months after the disease onset in a noteworthy proportion (approximately 3 out of 10) of patients with paucisymptomatic-to-mild clinical presentation deserves further investigations due to its possible pathophysiological implications and impact on the quality of life.The online version contains supplementary material available at 10.1186/s12879-022-07052-8. The prevalence, the persistence and the recovery rate of olfactory and gustatory dysfunctions were expressed as percentage with 95% confidence interval (CI) using Clopper-Pearson method .χ2) and the odds ratio (ORs) with 95% CIs. Differences in continuous variables were tested with Student’s t test for normally distributed ones, or the Mann–Whitney U test when variables showed a non-normal distribution.The association between self-reported presence of sensory alterations at baseline and putatively related variables in the study group was assessed computing the chi-square test , clinical characteristics  and clinical presentation (paucisymptomatic vs. mild) were independently associated with baseline sensory dysfunctions and complete recovery.Moreover, Kaplan–Meier estimates were reported to show the rates of complete recovery from the onset of the smell and/or taste loss .p ≤ 0.05.The level of statistical significance was set at Analysis was conducted with STATA/SE 15.0.From March 1st to June 16th, 2020, a total of 1175 COVID-19 confirmed cases were notified in the Foggia district, of which 653  were home-quarantined. Clinical presentation of COVID-19 was classified as paucisymptomatic in 55.6% (n = 363) of cases and mild in 19.1% (n = 125) of cases; 165 (25.3%) cases were classified as asymptomatic  reported at least one sensory dysfunction. In particular, 36.1%  reported anosmia, 37.7%  reported ageusia and 32.6%  reported both sensory dysfunctions  and related to a slower viral clearance  agreed to participate in the follow-up telephone survey (23 [11.4%] dropouts).After 12 months, 53 cases still reported partial chemosensory dysfunctions . Particularly, loss of smell persisted in 25.8%  and loss of taste in 21.3%  of cases; dysfunctions of both olfactory and gustatory sense were reported in 17.4%  of cases  of the respondents in the follow-up interview. The mean duration was 116.6 ± 150.6 days  for olfactory dysfunction and 104.4 ± 142.3 days  for gustatory dysfunction. No correlation was observed between the mean duration of chemosensory symptoms and any baseline patients’ demographic and clinical characteristics or the clinical presentation  in the follow-up reported complete resolution of the olfactory and gustatory disorders after 12 months, a substantial proportion of cases (approximately 3 out of 10) was still complaining of these symptoms. This proportion is remarkable, mainly because our data refer to a population with mild COVID-19. Few other studies comparable to ours in terms of sample size, demographic or clinical characteristics, adherence rates to the follow-up phase, have exceeded a 6-month follow-up and have investigated the recovery of olfactory and gustatory functions beyond this period , 28. Of Most available studies used online questionnaires or telephone interviews for the follow-up phase to collect general and specific clinical information on chemosensory disorders. As opposed to other studies , 28, we The rate of persistence of self-reported olfactory and gustatory dysfunctions observed in our study (29.8%) falls within the range reported by available studies (16 to 48%). In particular, Nguyen et al.  found thThe reason for the differences between the studies are still unclear and might be of various nature. Primarily, the estimated prevalence may be affected by the methodology used for the data collection. Indeed, it has been proven that the proportion of sensory dysfunctions is affected by the detection instruments, being higher with the use of objective measurements , 50. On Two very recent case–control studies have evaluated the persistence of smell and taste disorders at least one year after the onset of COVID-19 using psychophysical tests , 52. MorWe did not find any baseline demographic or clinical predictive factors for the persistence of symptoms. Interestingly, the occurrence of the syndrome defined “Long COVID” has been reported independently of the severity of the acute phase, being present also in patients with a mild disease , 38, 39.The recovery time is putatively dependent on the underlying pathophysiological mechanisms of sensory dysfunctions. These mechanisms were not investigated in our study but can hopefully be clarified with further purposely designed researches. In our analysis, patients were also asked to estimate the approximate duration of their symptoms. It emerged that a complete recovery of smell and taste tended to occur mostly in the first month after the acute phase of COVID-19. This is in agreement with Nguyen et al. , who docOlfactory and gustatory dysfunctions are generally considered less disabling and life-threatening than other sensory impairments. Thus, they may not receive adequate medical attention despite their impact on quality of life, especially if prolonged over months. However, olfactory and gustatory deprivations of various cause have been documented as disruptive on daily activities already before the spread of SARS-CoV-2 , 40, andOur study has some limitations. The self-reported assessment of the severity of symptoms and the retrospective investigation of their persistence within the 12-months follow-up period could be inaccurate due to a recall bias. Moreover, we did not record any possible pharmacological or rehabilitative intervention that could potentially affect the evolution of the olfactory and gustatory dysfunctions during the study period. As previously discussed, another possible limitation lies in the fact that, in our study, chemosensory dysfunctions were not assessed using objective examinations, such as psychophysical and electrophysiological tests, but only on a subjective basis. Therefore, underreporting of chemosensory dysfunctions cannot be excluded. However, in the perspective of a clinical approach taking into account the quality of life, a subjective evaluation may be more significant than an objective assessment of the disorders. Moreover, due to the absence of a control group, our findings could have been biased by the underlying prevalence of chemosensory dysfunctions in the general population , 54. AlsOlfactory and gustatory dysfunctions persist in a substantial portion of patients with previous paucisymptomatic-to-mild clinical COVID-19 up to 12 months after disease onset. As SARS-CoV-2 infection may be associated with long-term smell and/or taste loss that may significantly impact the quality of life, there is a need for a better understanding of the extent and duration of these symptoms. In addition, a better understanding of the physiological mechanisms driving the recovery is required to facilitate the development of effective preventive and therapeutic strategies.Additional file 1: Univariate analysis of variables associated with self-reported chemosensory dysfunctions in COVID-19 cases. District of Foggia , March 1st - June 16th, 2020.Additional file 2: Multivariate analysis of variables associated with self-reported chemosensory dysfunctions in COVID-19 cases. District of Foggia , March 1st - June 16th, 2020.Additional file 3: Multivariate analysis of variables associated with complete recovery of self-reported olfactory and gustatory dysfunctions in COVID-19 cases. District of Foggia , March 1st, 2020–June 16th, 2021.