Identification of a strong genetic risk factor for major depressive disorder in the human virome.

The heritability of major depressive disorder (MDD) is reportedly 30-50%. However, the genetic basis of its heritability remains unknown. Within SITH-1, a risk factor for MDD in human herpesvirus 6B (HHV-6B), we discovered a gene polymorphism with a large odds ratio for an association with MDD. It was a sequence whose number of repeats was inversely correlated with SITH-1 expression. This number was significantly lower in MDD patients. Rates for 17 or fewer repeats of the sequence were 67.9% for MDD and 28.6% for normal controls, with an odds ratio of 5.28. For patients with 17 or less repeats, the rate for presence of another MDD patient in their families was 47.4%, whereas there were no MDD patients in the families of patients with more than 17 repeats. Since HHV-6B is transmitted primarily mother to child and within families and persists for life, this gene polymorphism could potentially influence heritability of MDD.

Relationship between human herpesvirus 6 infection and inflammatory bowel disease using novel biomarker.

Objective: Inflammatory bowel disease (IBD) is closely related to stress and fatigue. Human herpesvirus 6B (HHV-6B) is reactivated by stress and fatigue and is associated with IBD. This study aimed to clarify the relationship between IBD and HHV-6B. Methods: Antibody titers to SITH-1, a protein specific to HHV-6B latent infection, were measured in 163 patients with IBD (107 with ulcerative colitis [UC] and 56 with Crohn's disease [CD]); clinical and endoscopic scores and depression scores of UC and CD were analyzed to examine the relationship between SITH-1 and IBD. The SITH-1 cut-off value was set as 1.96, according to known reports. Results: In patients with UC, C-reactive protein (CRP) level was significantly higher (1.5 vs 0.6 mg/L, P = 0.006) and disease exacerbation within 6 months after entry was significantly more common in the SITH-1 (+) group (20% vs 0%, P < 0.001). In the subanalysis comparing with and without UC exacerbation, the optimal cut-off value for SITH-1 to detect UC exacerbation was 3.44 (area under the curve: 0.81; 95% confidence interval: 0.72-0.90). CRP levels, SITH-1 levels, and disease activity scores by the clinical or endoscopic index were significantly higher in the exacerbation group than in the non-exacerbation group (2.6 vs 0.9 mg/L, P = 0.03; 4.90 vs 1.71, P < 0.001; 4 vs 3, P = 0.03; 5 vs 3, P = 0.02; respectively). Conclusion: Patients with UC with high titers of SITH-1 have high disease activity and frequent disease exacerbation. SITH-1 can be associated with UC disease activity.

SARS-CoV-2 S1 protein causes brain inflammation by reducing intracerebral acetylcholine production.

Neurological complications that occur in SARS-CoV-2 infection, such as olfactory dysfunction, brain inflammation, malaise, and depressive symptoms, are thought to contribute to long COVID. However, in autopsies of patients who have died from COVID-19, there is normally no direct evidence that central nervous system damage is due to proliferation of SARS-CoV-2. For this reason, many aspects of the pathogenesis mechanisms of such symptoms remain unknown. Expressing SARS-CoV-2 S1 protein in the nasal cavity of mice was associated with increased apoptosis of the olfactory system and decreased intracerebral acetylcholine production. The decrease in acetylcholine production was associated with brain inflammation, malaise, depressive clinical signs, and decreased expression of the cytokine degrading factor ZFP36. Administering the cholinesterase inhibitor donepezil to the mice improved brain inflammation, malaise and depressive clinical signs. These findings could contribute to the elucidation of the pathogenesis mechanisms of neurological complications associated with COVID-19 and long COVID.

VP26, a herpes simplex virus type 1 capsid protein, increases DNA methylation in COASY promoter region.

It has been reported that some specific changes in DNA methylation can be due to aging or infection by tumor-related viruses but the effect of herpes simplex virus type 1 (HSV-1) in this regard is unknown. HSV-1 is a well-known virus that causes cold sores. After the primary infection, the virus switches to latent infection and remains in the body for the whole life. As the location of DNA methylation, we focused on the promoter region of the COASY gene, which codes for coenzyme A synthase, because methylation in this region is reportedly associated with Alzheimer's disease (AD). During HSV-1 lytic infection, compared to non-infected cells, COASY DNA methylation decreased but when HSV-1 replication was inhibited by acyclovir, an anti-herpes agent, COASY DNA methylation increased. In addition, for expression of immediate early protein only, there was no significant change in COASY DNA methylation, while for expression of the capsid protein VP26, a late protein known to bind with DNA methyltransferase DNMT3A, in the nucleus only, COASY DNA methylation significantly increased compared to the control, without changes in DNMT3A mRNA. Our results suggested that DNA methylation occurred not due to transcriptional changes in DNMT3A but through translational regulation. In this research, we showed that host COASY DNA methylation is altered by HSV-1 infection, in particular by HSV-1 VP26. It is a potential cause of various diseases, and this is particularly relevant for AD.

Blood DNA Methylation Levels in the WNT5A Gene Promoter Region: A Potential Biomarker for Agitation in Subjects with Dementia.

BACKGROUND: Behavioral and psychological symptoms of dementia (BPSD) cause a heavy burden for both patient and caregivers. These symptoms are diverse, and their mechanism is still unclear. Agitation is the most common and difficult to treat among BPSD. In recent years, while changes in DNA methylation levels have been receiving attention as a biomarker of aging and dementia, associations with BPSD have not been examined. OBJECTIVE: Focusing on agitation, the objective of the present study was to identify a region where changes in DNA methylation levels are associated with agitation. METHODS: Using genome-wide DNA methylation analysis data for 7 dementia subjects with agitation, 5 dementia subjects without agitation, and 4 normal elderly controls, we determined a signaling pathway in the WNT5A gene promoter region to be associated with agitation. Based on this result, we measured DNA methylation levels in this region for 26 dementia subjects with agitation and 82 dementia subjects without agitation by means of methylation-sensitive high-resolution melting (MS-HRM) analysis. RESULTS: The WNT5A DNA methylation level in dementia subjects with agitation was significantly lower than in those without agitation (p = 0.001). Changes in WNT5A DNA methylation levels were not influenced by age, sex, body mass index, APOE ɛ4, medication, or inflammatory cytokines. CONCLUSION: Our results suggested an association of agitation with Wnt signaling, in particular with changes in WNT5A DNA methylation levels, which could be a potentially useful biomarker for predicting the appearance of agitation. It may contribute to the elucidation of the mechanism of BPSD.

Cooperative activation of the human herpesvirus 6B U79/80 early gene promoter by immediate-early proteins IE1B and IE2B.

The human herpesvirus 6B (HHV-6B) U79/80 gene belongs to the early gene class and appears as early as 3 hr postinfection. It is one of the most abundantly expressed transcripts and a useful diagnostic marker for viral reactivation. However, the expression mechanisms of the U79/80 gene remain unclear. To identify the viral factor(s) that activates the U79/80 promoter along with other HHV-6B core early gene promoters, p41, DNA polymerase, and U41, we examined the activities of U79/80 and other early gene promoters. In HHV-6B-infected MT-4 cells, U79/80 promoter activity was the highest among early gene promoters. In addition, we identified that HHV-6B immediate-early (IE)2B protein is one of the viral proteins involved in the activation of the U79/80 and other early gene promoters. Although the IE2B could independently activate these early gene promoters, the presence of IE1B significantly augmented the activities of early gene promoters. We also found that IE2B bound three human cytomegalovirus IE2-binding consensus, cis repression signal (CRS), within the U79/80 promoter. Moreover, the U79/80 promoter was activated by cellular factors, which are highly expressed in MT-4 cells, instead of HeLa cells because it was upregulated by mock infection and in the absence of IE2B. These results suggested that the activation mechanism of the U79/80 gene differs from other HHV-6B core early genes, apparently supporting its rapid and abundant expression. Therefore, the U79/80 early gene is an actually suitable biomarker of HHV-6B reactivation.

Increased blood COASY DNA methylation levels a potential biomarker for early pathology of Alzheimer's disease.

Early diagnosis of dementia including Alzheimer's disease (AD) is an urgent medical and welfare issue. However, to date, no simple biometrics have been available. We reported that blood DNA methylation levels of the COASY gene, which encodes coenzyme A synthase, were increased in individuals with AD and amnestic mild cognitive impairment (aMCI). The present study sought to replicate these findings with larger numbers of samples. Another objective was to clarify whether COASY methylation is associated with neurodegeneration through a comparison of AD, AD with cardiovascular disease (CVD), and vascular dementia (VaD). We measured blood COASY methylation levels in normal controls (NCs) (n = 200), and individuals with aMCI (n = 22), AD (n = 151), and VaD (n = 21). Compared with NCs, they were significantly higher in individuals with aMCI and AD. Further, they were significantly higher in AD patients without cardiovascular diseases compared to AD patients with them. These findings suggest that COASY methylation levels may be related to neurodegeneration in AD.

Human Herpesvirus 6B Greatly Increases Risk of Depression by Activating Hypothalamic-Pituitary -Adrenal Axis during Latent Phase of Infection.

Little is known about the effect of latent-phase herpesviruses on their host. Human herpesvirus 6B (HHV-6B) is one of the most ubiquitous herpesviruses, and olfactory astrocytes are one of the most important sites of its latency. Here, we identified SITH-1, an HHV-6B latent protein specifically expressed in astrocytes. Mice induced to produce SITH-1 in their olfactory astrocytes exhibited olfactory bulb apoptosis, a hyper-activated hypothalamic-pituitary-adrenal (HPA) axis and depressive symptoms. The binding of SITH-1 to the host protein calcium-modulating ligand (CAML) to form an activated complex promoted the influx of extracellular calcium. The serum antibody titers for depressive patients with respect to this activated complex were significantly higher than for normal controls (p = 1.78 × 10-15), when the antibody positive rates were 79.8% and 24.4%, respectively, and the odds ratio was 12.2. These results suggest that, in the latent phase, HHV-6B may be involved in the onset of depression.

Classification and Prognostic Stratification of Bronchopulmonary Neuroendocrine Neoplasms.

The accuracy and reproducibility of the World Health Organization (WHO) 2015 classification of bronchopulmonary neuroendocrine neoplasms (BP-NENs) is disputed. The aim of this study is to classify and grade BP-NENs using the WHO 2019 classification of digestive system NENs (DiS-NEN-WHO 2019), and to analyze its accuracy and prognostic impact. Two BP-NEN cohorts from Japan and Germany, 393 tumors (88% surgically resected), were reviewed and the clinicopathological data of the resected tumors (n = 301) correlated to patients' disease-free survival (DFS). The DiS-NEN-WHO 2019 stratified the 350 tumors into 91 (26%) neuroendocrine tumors (NET) G1, 52 (15%) NET G2, 15 (4%) NET G3, and 192 (55%) neuroendocrine carcinomas (NEC). NECs, but not NETs, were immunohistochemically characterized by abnormal p53 (100%) and retinoblastoma 1 (83%) expression. The Ki67 index, which was on average 4 times higher than mitotic count (p < 0.0001), was prognostically more accurate than the mitotic count. NET G3 patients had a worse outcome than NET G1 (p < 0.01) and NET G2 patients (p = 0.02), respectively. No prognostic difference was detected between NET G3 and NEC patients after 5 year DFS. It is concluded that stratifying BP-NEN patients according to the DiS-NEN-WHO 2019 classification results in 3 prognostically well-defined NET groups, if grading is solely based on Ki67 index. Mitotic count alone may underestimate malignant potential of NETs.

Clinicopathological Profiling of Lung Carcinoids with a Ki67 Index > 20.

The clinicopathological features of lung neuroendocrine neoplasms (NEN) with a high proliferative index at the border area between atypical carcinoid and neuroendocrine carcinoma have not been investigated so far. The aim of this study was, therefore, to search for lung NENs which are well differentiated but show Ki67 values that overlap with those of poorly differentiated (PD)-NENs. Resected lung NENs from 244 Japanese patients were reviewed, and Ki67 indices were assessed in all tumors. The data were then correlated to clinicopathological parameters and patient outcome. Among 59 (24%) well-differentiated (WD)-NENs and 185 (76%) lung PD-NENs, 7 were defined as WD-NENs with Ki67 indices > 20%. The Ki67 indices of these tumors (mean 29%, range 24-36) were significantly lower than those of PD-NENs (mean 74%, range 34-99). All WD-NENs with Ki67 > 20% lacked abnormal p53 and loss of retinoblastoma 1 (Rb1) expression. In contrast, many PD-NENs expressed p53 (48%) and showed loss of Rb1 (86%). The 2- and 5-year disease-free survival rates in WD-NEN patients with Ki67 > 20% were lower than those of WD-NEN patients with Ki67 ≤20% (p < 0.01 for disease-free and overall survival). No statistical differences were detected between outcome of WD-NEN patients with Ki67 > 20% and those of PD-NEN. It is concluded that WD-NEN patients with Ki67 > 20% share the morphological and immunohistochemical features of WD-NEN patients with Ki67 ≤20%, but they have a worse prognosis, suggesting that this tumor group requires particular attention in future classifications and probably new therapeutic regimes.

Metastasis of adenoid cystic carcinoma from a submandibular gland to the larynx.

The present article discusses the first reported case of adenoid cystic carcinoma (ACC) metastasis from a submandibular gland to the larynx. Both treatments of distant metastasis of ACC and secondary laryngeal tumor are challenging. Despite its slow progression, ACC is associated with high rates of local recurrence, distant metastasis, and poor prognosis. Patients with secondary laryngeal cancer often have other concurrent metastatic lesions. Therefore, treatment selection should consider the biological behavior of the tumor and characteristics of the laryngeal lesion, along with the general condition and quality of life of the patient. The patient (55-year-old female) had a history of ACC of the right submandibular gland, removed surgically 9 years prior to the present consultation. Follow-up showed multiple pulmonary metastases. The patient complained of dysphonia lasting 3 months. Following the diagnosis of ACC metastasis to the larynx (supraglottic) and a neck lymph node via biopsy, we performed partial laryngectomy, left neck dissection, and tracheotomy. Histopathological examination showed an increase in the tumor grade over time. Two months after discharge, there was no obvious local recurrence or increase in lung metastasis.

Inflammation and PD-L1 expression in pulmonary neuroendocrine tumors.

In the light of novel cancer immune therapies, the status of antitumor inflammatory response and its regulation has gained much attention in patients with lung cancer. Ample datasets exist for non-small-cell lung cancer, but those for pulmonary neuroendocrine tumors are scarce and controversial. Here, tumor-associated inflammation, CD8+ cell infiltration and PD-L1 status were evaluated in a cohort of 57 resected carcinoids and 185 resected neuroendocrine carcinomas of the lung (58 large cell carcinomas and 127 small cell carcinomas). Data were correlated with clinicopathological factors and survival. Moderate or high tumor-associated inflammation was detected in 4 carcinoids (7%) and in 37 neuroendocrine carcinomas (20%). PD-L1 immunoreactivity was seen in immune cells of 73 (39%) neuroendocrine carcinomas, while tumor cells were labeled in 21 (11%) cases. Inflammatory cells and tumor cells in carcinoids lacked any PD-L1 expression. In neuroendocrine carcinomas, PD-L1 positivity in immune cells, but not in tumor cells, was associated with intratumoral CD8+ cell infiltration (P < 0.001), as well as with the severity of tumor-associated inflammation (P < 0.001). In neuroendocrine carcinomas, tumor-associated inflammation and PD-L1 positivity in immune cells correlated with prolonged survival and the latter factor was also an independent prognosticator (P < 0.01, hazard ratio 0.4 for overall survival, P < 0.001 hazard ratio 0.4 for disease-free survival). Taken together, in neuroendocrine tumors, antitumor inflammatory response and PD-L1 expression are largely restricted to neuroendocrine carcinomas, and in this tumor entity, PD-L1 expression in inflammatory cells is positively correlated to patient survival.

Increased interleukin-1ß and basic fibroblast growth factor levels in the cerebrospinal fluid during human herpesvirus-6B (HHV-6B) encephalitis.

Human herpesvirus 6B (HHV-6B) causes exanthema subitum in infants and is known to be mildly pathogenic. However, HHV-6B infection can induce febrile seizures in a high percentage of patients, and in rare cases, result in encephalitis. We detected higher levels of interleukin (IL)-1ß and basic fibroblast growth factor (bFGF) in the cerebrospinal fluid (CFS) of patients with HHV-6B encephalitis when compared to those in patients with non-HHV-6B-induced febrile seizures. In vitro, IL-1ß and bFGF enhanced HHV-6B gene expression in infected U373 astrocytes during the initial and maintenance phases of infection, respectively. These findings indicated that IL-1ß and bFGF contribute to HHV-6B growth and the onset of encephalitis.

SOX2 and Rb1 in esophageal small-cell carcinoma: their possible involvement in pathogenesis.

Clinicopathological features and pathogenesis of esophageal small-cell carcinoma remain unclear. We hypothesized common cellular origin and pathogenesis in small-cell carcinoma of esophagus and lung associated with SOX2 overexpression and loss of Rb1. Expression of squamous-basal markers (CK5/6 and p40), glandular markers (CK18 and CEA), SOX2, and Rb1 were evaluated in 15 esophageal small-cell carcinomas, 46 poorly differentiated squamous cell carcinomas, and 88 small-cell lung carcinoma, as well as 16 embryonic esophagus. Esophageal small-cell carcinoma expressed higher levels of glandular markers and lower levels of squamous-basal markers than poorly differentiated squamous cell carcinoma. No significant differences were observed in immunohistochemistry profiles between small-cell carcinoma of the esophagus and the lung. SOX2 expression was high in esophageal small-cell carcinoma (70%±33% of nuclei), small-cell lung carcinoma (70%±26%), and the embryonic esophagus (75%±4%), and it was significantly lower in poorly differentiated squamous cell carcinoma (29%±28%). Rb1 expression was significantly lower in esophageal small-cell carcinoma (0.3%±1%), small-cell lung carcinoma (2%±6%), and the embryonic esophagus (7%±5%), and it was significantly higher in poorly differentiated squamous cell carcinoma (51%±24%). The immunohistochemistry profiles of small-cell carcinoma of the esophagus and the lung are highly similar. The loss of Rb1 function is a key contributor to the pathogenesis of both neoplasms. In addition, SOX2 overexpression observed in esophageal and lung small-cell carcinoma as well as in the embryonic esophagus indicated that esophageal small-cell carcinoma may arise from embryonic-like stem cells in the esophageal epithelium. The two distinct differentiation patterns (neuroendocrine and glandular) of esophageal small-cell carcinoma further support the fact that SOX2 has a pivotal role in the differentiation of pluripotent stem cells into esophageal small-cell carcinoma cells.

Human herpesvirus 6 and 7 are biomarkers for fatigue, which distinguish between physiological fatigue and pathological fatigue.

Fatigue reduces productivity and is a risk factor for lifestyle diseases and mental disorders. Everyone experiences physiological fatigue and recovers with rest. Pathological fatigue, however, greatly reduces quality of life and requires therapeutic interventions. It is therefore necessary to distinguish between the two but there has been no biomarker for this. We report on the measurement of salivary human herpesvirus (HHV-) 6 and HHV-7 as biomarkers for quantifying physiological fatigue. They increased with military training and work and rapidly decreased with rest. Our results suggested that macrophage activation and differentiation were necessary for virus reactivation. However, HHV-6 and HHV-7 did not increase in obstructive sleep apnea syndrome (OSAS), chronic fatigue syndrome (CFS) and major depressive disorder (MDD), which are thought to cause pathological fatigue. Thus, HHV-6 and HHV-7 would be useful biomarkers for distinguishing between physiological and pathological fatigue. Our findings suggest a fundamentally new approach to evaluating fatigue and preventing fatigue-related diseases.

Cytokeratin 7-positive/cytokeratin 20-negative cecal adenocarcinoma metastatic to the uterine cervix: a case report.

BACKGROUND: The vast majority of uterine cervical malignancies are primary carcinomas, and secondary neoplasms that metastasize to the uterine cervix from a distant organ are uncommon. Although relatively rare, metastases to the uterine cervix from a primary colon cancer have been reported. We report a rare case of metastatic carcinoma originating from a cecal adenocarcinoma with an unusual cytokeratin 7/cytokeratin 20 immunophenotype. CASE PRESENTATION: A 74-year-old postmenopausal Japanese woman was referred to our hospital for the evaluation of a uterine tumor. She had a past medical history of cecal cancer and had undergone laparoscopically assisted right hemicolectomy at the age of 69 years. During follow-up, she was found to have elevated levels of the tumor markers carbohydrate antigen 19-9 (179.7 IU/mL) and carcinoembryonic antigen (26.9 µg/L). Positron emission tomography/computed tomography showed a focus of high 18F-fluorodeoxyglucose uptake in her uterus. Examination of a cervical biopsy found a poorly differentiated adenocarcinoma that was immunopositive for cytokeratin (CK)7 and caudal-related homeobox 2 (CDX2) expression and immunonegative for cytokeratin 20 expression. The patient underwent radical hysterectomy and bilateral salpingo-oophorectomy. Histopathological examination found invasive growth of irregular and atypical ductal hyperplasia. Immunohistochemical staining of the tumor specimen revealed the same immunophenotype as the biopsy specimen. The cecal cancer specimen from her previous surgery was also examined and found to have the same immunophenotype. The histopathological diagnosis was cecal adenocarcinoma metastatic to the uterine cervix. The patient is currently receiving adjuvant chemotherapy and to date is without evidence of recurrent disease. CONCLUSIONS: Our report illustrates the importance of immunohistochemistry for the correct diagnosis of the origin of a uterine cervical adenocarcinoma in a patient with a medical history of colorectal cancer. Re-examination of a previous oncological specimen is critical for cases with a uterine lesion that is difficult to identify as primary or metastatic cancer.

Expression, purification, and immobilization of recombinant tamavidin 2 fusion proteins.

Tamavidin 2 is a fungal avidin-like protein that binds biotin with high affinity. Unlike avidin or streptavidin, tamavidin 2 in soluble form is produced at high levels in Escherichia coli. In this chapter, we describe a method for immobilization and purification of recombinant proteins with the use of tamavidin 2 as an affinity tag. The protein fused to tamavidin 2 is tightly immobilized and simultaneously purified on biotinylated magnetic microbeads without loss of activity.

Tamavidin 2-HOT, a highly thermostable biotin-binding protein.

Tamavidin 2 is a fungal tetrameric protein that binds with high affinity to biotin, like avidin and streptavidin. We replaced asparagine-115, which lies in a subunit-subunit interface of tamavidin 2, with cysteine to generate the novel, highly thermostable protein tamavidin 2-HOT. Tamavidin 2-HOT retained more than 80% of its biotin-binding activity even after incubation at 99.9°C for 60min and was fully active in 70% dimethylsulfoxide for 30min, whereas in these harsh conditions, avidin, streptavidin, and tamavidin 2 lost their activities (less than 20% of their biotin-binding activities). The Tm in which the biotin-binding activity becomes half of tamavidin 2-HOT was 105°C, at least 20°C higher than those of avidin, streptavidin, and tamavidin 2. Because a reducing agent removed the thermal stability of tamavidin 2-HOT, the N115C mutation likely created disulfide bridges that stabilized inter-subunit associations. Tamavidin 2-HOT is efficiently produced in the soluble form by Escherichia coli for practical use. The isoelectric point of tamavidin 2-HOT (7.4) is sufficiently low to reduce the chance for non-specific binding of non-target molecules due to high positive charges. Therefore, tamavidin 2-HOT may be useful in diverse novel applications that take advantage of its high biotin-binding capability that can withstand harsh conditions.

Increase in the IgG avidity index due to herpes simplex virus type 1 reactivation and its relationship with cognitive function in amnestic mild cognitive impairment and Alzheimer's disease.

After infection with herpes simplex virus type 1 (HSV-1), latent infection persists for life in the trigeminal ganglion and reactivation results in an outbreak of cold sores around the mouth. Many previous studies have reported HSV-1 reactivation to be a risk factor for Alzheimer's disease (AD). This study enrolled subjects with AD (n=85), subjects with amnestic mild cognitive impairment (aMCI; a prodromal stage of AD) (n=34), and healthy controls (n=28). The avidity index of anti-HSV-1 IgG antibodies--a known indicator of HSV-1 reactivation--was measured in order to clarify the relationship between HSV-1 reactivation and symptoms of cognitive function in AD. Cognitive function in AD and aMCI were evaluated using scores from the mini-mental state examination (MMSE) and frontal assessment battery (FAB). The results showed that the subjects with aMCI, for which cerebral function is better preserved than subjects with AD, had a higher anti-HSV-1 IgG antibody avidity index than the AD subjects or healthy controls. Furthermore, the anti-HSV-1 IgG antibody avidity index was even higher in the subjects with high MMSE scores on orientation to time and three-step command subscores. We observed a negative correlation between the anti-HSV-1 IgG antibody avidity index and plasma BDNF concentration, which is an indicator of encephalitis. This suggests that HSV-1 reactivation, as observed through an increase in the anti-HSV-1 IgG avidity index, does not progress to encephalitis. These results suggest that HSV-1 reactivation occurs from the stage of aMCI, which is prodromal to AD, and can affect AD symptoms without an intermediary stage of severe encephalitis. The study demonstrates that the anti-HSV-1 IgG antibody avidity index could be a useful biomarker for the early diagnosis of aMCI as well as AD, and suggests that antiviral medication to treat HSV-1 could play a role in preventing the onset of AD.

Engineering of novel tamavidin 2 muteins with lowered isoelectric points and lowered non-specific binding properties.

The avidin-biotin interaction is widely employed as a universal tool in numerous biotechnological applications. In avidin-biotin technology, non-specific binding to biological macromolecules is a hindrance. The major origin of this non-specific binding is the electrical charge of the surface of biotin-binding proteins. Tamavidin 2, a fungal avidin-like protein that binds biotin with an extremely high affinity, can be produced as a soluble recombinant protein in Escherichia coli. The isoelectric point of tamavidin 2 is 7.4-7.5, lower than avidin (10.0), and slightly higher than that of streptavidin (6.0-7.5). Here, we genetically engineered charge mutants of tamavidin 2 to reduce non-specific binding. By substituting an acidic residue (glutamic acid) for basic residues (arginine and lysine), we constructed three mutant proteins (muteins) and confirmed their high-level production in soluble form in E. coli, as well as that of tamavidin 2. We then tested these proteins for non-specific binding to salmon sperm DNA, glycoproteins (integrin and fibronectin), and IgG from human sera. The muteins showed lower non-specific binding than tamavidin 2 to these macromolecules. In particular, one mutein, tamavidin-R104EK141E, which had the lowest isoelectric point (5.8-6.2) among avidin, streptavidin and tamavidin 2, displayed the lowest non-specific binding. The affinity of this mutein to biotin was high, comparable with that of tamavidin 2. These findings indicate that tamavidin-R104EK141E has the potential to serve as a robust tool in the numerous applications of biotin-binding proteins.