Berberine Metabolites Stimulate GLP-1 Secretion by Alleviating Oxidative Stress and Mitochondrial Dysfunction.

Berberine (BBR) is a principal component of Rhizoma coptidis known for its therapeutic potential in treating diseases such as type 2 diabetes mellitus (T2DM) and obesity. Despite the trace levels of BBR in plasma, it's believed that its metabolites play a pivotal role in its biological activities. While BBR is recognized to promote GLP-1 production in intestinal L cells, the cytoprotective effects of its metabolites on these cells are yet to be explored. The present study investigates the effects of BBR metabolites on GLP-1 secretion and the underlying mechanisms. Our results revealed that, out of six BBR metabolites, berberrubine (BBB) and palmatine (PMT) significantly increased the production and glucose-stimulated secretion of GLP-1 in GLUTag cells. Notably, both BBB and PMT could facilitate GLP-1 and insulin secretion and enhance glucose tolerance in standard mice. Moreover, a single dose of PMT could markedly increase plasma GLP-1 and improve glucose tolerance in mice with obesity induced by a high-fat diet. In palmitic acid or TNF[Formula: see text]-treated GLUTag cells, BBB and PMT alleviated cell death, oxidative stress, and mitochondrial dysfunction. Furthermore, they could effectively reverse inflammation-induced inhibition of the Akt signaling pathway. In general, these insights suggest that the beneficial effects of orally administered BBR on GLP-1 secretion are largely attributed to the pharmacological activity of BBB and PMT by their above cytoprotective effects on L cells, which provide important ideas for stimulating GLP-1 secretion and the treatment of T2DM.

Comparative analysis of primate and pig cells reveals primate-specific PINK1 expression and phosphorylation.

PTEN-induced putative kinase 1 (PINK1), a mitochondrial kinase that phosphorylates Parkin and other proteins, plays a crucial role in mitophagy and protection against neurodegeneration. Mutations in PINK1 and Parkin can lead to loss of function and early onset Parkinson's disease. However, there is a lack of strong in vivo evidence in rodent models to support the theory that loss of PINK1 affects mitophagy and induces neurodegeneration. Additionally, PINK1 knockout pigs ( Sus scrofa) do not appear to exhibit neurodegeneration. In our recent work involving non-human primates, we found that PINK1 is selectively expressed in primate brains, while absent in rodent brains. To extend this to other species, we used multiple antibodies to examine the expression of PINK1 in pig tissues. In contrast to tissues from cynomolgus monkeys ( Macaca fascicularis), our data did not convincingly demonstrate detectable PINK1 expression in pig tissues. Knockdown of PINK1 in cultured pig cells did not result in altered Parkin and BAD phosphorylation, as observed in cultured monkey cells. A comparison of monkey and pig striatum revealed more PINK1-phosphorylated substrates in the monkey brain. Consistently, PINK1 knockout in pigs did not lead to obvious changes in the phosphorylation of Parkin and BAD. These findings provide new evidence that PINK1 expression is specific to primates, underscoring the importance of non-human primates in investigating PINK1 function and pathology related to PINK1 deficiency.

Analysis of Pediatric Surgical Disease Spectrum in a Tertiary Hospital.

Objective: To analyze the main disease composition of children hospitalized in pediatric surgery, explore the correlation between disease types and gender, and provide a reference for hospital management and pediatric disease prevention. Methods: Using ICD-10 codes as the classification standard for disease diagnosis, a statistical analysis was conducted on the disease composition of children hospitalized in the Pediatric Surgery Department of the Second Affiliated Hospital of Xi'an Jiaotong University from January 1, 2015, to December 31, 2015, followed by the establishment of a clinical database. A total of 1647 male patients and 817 female patients were enrolled in the study, resulting in a male-to-female ratio of 2:1. The age range of the patients spanned from 0 to 18 years, with a marked imbalance in patient distribution among the various age groups. Statistical analysis was conducted using SPSS version 18.0 software. A chi-square test was performed to analyze the differences in the composition of disease systems and the composition of major diseases in terms of sex and age. Results: Pediatric patients were admitted with complex and diverse diseases in 2015, involving 15 systems of the human body and 400 diseases. Digestive system diseases, tumors, congenital malformations, and genitourinary system diseases were the top four diseases accounting for 83.5% of all pediatric cases. 561 patients were aged 0 years, accounting for 22.3% of all cases, while 1,801 patients fell within the 0-5 years age group, constituting 73.1% of the total. The differences in disease system composition among different sex and age groups of pediatric surgical inpatients were statistically significant (P = .001). There are statistically significant differences in the length of hospital stay and hospitalization costs among pediatric surgical inpatients in different age groups (P = .001). Conclusion: To strengthen the diagnosis and treatment of pediatric surgical diseases, we should strengthen the construction of key departments, optimize the consultation process according to the characteristics of children's disease spectrum, and improve the level of diagnosis and treatment of pediatric surgical diseases.

[Effect of breastfeeding on immune function in infants with human cytomegalovirus infection]. / æ¯ä¹³å–‚å »å¯¹äººå·¨ç»†èƒžç— æ¯’æ„ŸæŸ“å©´å„¿å ç–«åŠŸèƒ½çš„å½±å“.

OBJECTIVES: To study the effect of breastfeeding on immune function in infants with human cytomegalovirus (HCMV) infection. METHODS: A retrospective analysis was performed on the medical data of 135 infants with HCMV infection who were admitted to Children's Hospital Affiliated to Zhengzhou University from January 2021 to May 2022, and all these infants received breastfeeding. According to the results of breast milk HCMV-DNA testing, the infants were divided into two groups: breast milk HCMV positive (n=78) and breast milk HCMV negative (n=57). According to the median breast milk HCMV-DNA load, the infants in the breast milk HCMV positive group were further divided into two subgroups: high viral load and low viral load (n=39 each). Related indicators were compared between the breast milk positive and negative HCMV groups and between the breast milk high viral load and low viral load subgroups, including the percentages of peripheral blood lymphocyte subsets (CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD19+ B cells), CD4+/CD8+ ratio, IgG, IgM, IgA, and urine HCMV-DNA load. RESULTS: There were no significant differences in the percentages of CD3+ T cells, CD3+CD4+ T cells, CD3+CD8+ T cells, and CD19+ B cells, CD4+/CD8+ ratio, IgG, IgM, IgA, and urine HCMV-DNA load between the breast milk HCMV positive and HCMV negative groups, as well as between the breast milk high viral load and low viral load subgroups (P>0.05). CONCLUSIONS: Breastfeeding with HCMV does not affect the immune function of infants with HCMV infection.

An approach combining deep learning and molecule docking for drug discovery of cathepsin L.

OBJECTIVES: Cathepsin L (CTSL) is a promising therapeutic target for metabolic disorders and COVID-19. However, there are still no clinically available CTSL inhibitors. Our objective is to develop an approach for the discovery of potential reversible covalent CTSL inhibitors. METHODS: The authors combined Chemprop, a deep learning-based strategy, and the Schrödinger CovDock algorithm to identify potential CTSL inhibitors. First, they used Chemprop to train a deep learning model capable of predicting whether a molecule would inhibit the activity of CTSL and performed predictions on ZINC20 in-stock librarie (~9.2 million molecules). Then, they selected the top-200 predicted molecules and performed the Schrödinger covalent docking algorithm to explore the binding patterns to CTSL (PDB: 5MQY). The authors then calculated the binding energies using Prime MM/GBSA and examined the stability between the best two molecules and CTSL using 100ns molecular dynamics simulations. RESULTS: The authors found five molecules that showed better docking results than the well-known cathepsin inhibitor odanacatib. Notably, two of these molecules, ZINC-35287427 and ZINC-1857528743, showed better docking results with CTSL compared to other cathepsins. CONCLUSION: Our approach enables drug discovery from large-scale databases with little computational consumption, which will save the cost and time required for drug discovery.

[Arthroscopic subscapularis augmentation for the treatment of recurrent dislocation of shoulder with severe glenoid injury].

OBJECTIVE: To investigate the method and clinical effects of the treatment of recurrent shoulder dislocation with severe glenoid injury by arthroscopic subscapularis augmentation. METHODS: From March 2019 to August 2020, 16 patients with recurrent dislocation of shoulder with severe glenoid injury underwent arthroscopic subscapularis augmentation, including 10 males and 6 females, aged from 18 to 50 years old with an average of (29.06±10.54) years old, 4 cases of left shoulder and 12 cases of right shoulder.Visual analogue scale (VAS), American Shoulder and Elbow Surgeons (ASES) score and Rowe score were used to evaluate shoulder function and stability before and after operation. RESULTS: All the 16 patients were followed up from 12 to 29 months, with an average of (18.75±7.26) months. VAS score decreased from 5.25±1.13 before operation to 1.37±0.65 at the final follow-up;ASES score increased from 59.44±9.93 before surgery to 90.88±4.00 at the final follow-up; Rowe score of shoulder increased from 51.56±8.89 before surgery to 92.19±7.06 at the final follow-up, and the differences were statistically significant (P<0.05). No dislocation was observed during follow-up. No clinical complications such as incision infection, vascular and nerve injury occurred in all patients after operation. CONCLUSION: Arthroscopic subscapularis augmentation in the treatment of recurrent dislocation of shoulder with severe glenoid injury is satisfactory. It is an effective treatment method of recurrent dislocation of shoulder joint with severe glenoid injury, and external rotation function in patients with almost unaffected.

A high TSH level is associated with diabetic macular edema: a cross-sectional study of patients with type 2 diabetes mellitus.

Aims: In this study, we determined the association between thyroid-stimulating hormone (TSH) and diabetic macular edema (DME) by assessing the prevalence and risk factors for DME in type 2 diabetes mellitus (T2DM) patients with different thyroid dysfunctions. Methods: This was a retrospective cross-sectional study including 1003 euthyroid and 92 subclinical hypothyroidism (SCH) T2DM patients. DME status was detected by optical coherence tomography (OCT). The association between TSH and DME and the impact of TSH on DME were analyzed. Results: The DME prevalence was 28.3% in the SCH patients and 14.0% in the euthyroid population. The serum FT4 (P = 0.001) and FT3 (P < 0.001) levels were significantly higher in the non-DME group than in the DME group, and the TSH level (P < 0.001) was significantly lower. Four subgroups (G1-G4) were divided by TSH level, and the chi-square test indicated that even in the normal range, the TSH level was positively related to DME prevalence (P = 0.001). Subgroup data indicated that the association between TSH and DME detected by OCT (P = 0.001) was stronger than the correlation between TSH and diabetic retinopathy detected by digital retinal photographs (P = 0.027). The logistic regression model confirmed that elevated TSH was an independent risk factor for DME. The odds ratio was 1.53 (P = 0.02). Conclusions: A high TSH level was an independent risk factor for DME. More attention should be given to the TSH level in T2DM patients due to its relationship with diabetic complications.

Potential drug discovery for COVID-19 treatment targeting Cathepsin L using a deep learning-based strategy.

Cathepsin L (CTSL), a cysteine protease that can cleave and activate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, could be a promising therapeutic target for coronavirus disease 2019 (COVID-19). However, there is still no clinically available CTSL inhibitor that can be used. Here, we applied Chemprop, a newly trained directed-message passing deep neural network approach, to identify small molecules and FDA-approved drugs that can block CTSL activity to expand the discovery of CTSL inhibitors for drug development and repurposing for COVID-19. We found 5 molecules (Mg-132, Z-FA-FMK, leupeptin hemisulfate, Mg-101 and calpeptin) that were able to significantly inhibit the activity of CTSL in the nanomolar range and inhibit the infection of both pseudotype and live SARS-CoV-2. Notably, we discovered that daptomycin, an FDA-approved antibiotic, has a prominent CTSL inhibitory effect and can inhibit SARS-CoV-2 pseudovirus infection. Further, molecular docking calculation showed stable and robust binding of these compounds with CTSL. In conclusion, this study suggested for the first time that Chemprop is ideally suited to predict additional inhibitors of enzymes and revealed the noteworthy strategy for screening novel molecules and drugs for the treatment of COVID-19 and other diseases with unmet needs.

The mechanism and effects of remdesivir-induced developmental toxicity in zebrafish: Blood flow dysfunction and behavioral alterations.

The antiviral drug remdesivir has been used to treat the growing number of coronavirus disease 2019 (COVID-19) patients. However, the drug is mainly excreted through urine and feces and introduced into the environment to affect non-target organisms, including fish, which has raised concerns about potential ecotoxicological effects on aquatic organisms. Moreover, studies on the ecological impacts of remdesivir on aquatic environments have not been reported. Here, we aimed to explore the toxicological impacts of microinjection of remdesivir on zebrafish early embryonic development and larvae and the associated mechanism. We found that 100 µM remdesivir delayed epiboly and impaired convergent movement of embryos during gastrulation, and dose-dependent increases in mortality and malformation were observed in remdesivir-treated embryos. Moreover, 10-100 µM remdesivir decreased blood flow and swimming velocity and altered the behavior of larvae. In terms of molecular mechanisms, 80 differentially expressed genes (DEGs) were identified by transcriptome analysis in the remdesivir-treated group. Some of these DEGs, such as manf, kif3a, hnf1ba, rgn, prkcz, egr1, fosab, nr4a1, and ptgs2b, were mainly involved in early embryonic development, neuronal developmental disorders, vascular disease and the blood flow pathway. These data reveal that remdesivir can impair early embryonic development, blood flow and behavior of zebrafish embryos/larvae, probably due to alterations at the transcriptome level. This study suggests that it is important to avoid the discharge of remdesivir to aquatic ecosystems and provides a theoretical foundation to hinder remdesivir-induced ecotoxicity to aquatic environments.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development.

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

Nifuroxazide improves insulin secretion and attenuates high glucose-induced inflammation and apoptosis in INS-1 cells.

Inflammation and oxidative stress are important factors that cause islet ß-cell dysfunction. STAT3 is not only a major factor in cell proliferation and differentiation, but also plays an important role in mediating inflammation. As a potent inhibitor of STAT3, the effect of Nifuroxazide (Nifu) on pancreatic islet cells in a high glucose environment has not been reported. In the present study, we used high concentration glucose-induced INS-1 cells to examine the effects of Nifu on high glucose-induced cell function by glucose-stimulated insulin secretion (GSIS). The effects of Nifu on high glucose-induced oxidative stress were recorded by oxidative factors and antioxidant factors. Simultaneously, the effect of Nifu on the inflammatory response, apoptosis, and STAT3/SOCS3 signal pathway were validated by quantitative real-time PCR (qRT-PCR) and Western blot. Our study indicated that Nifu significantly improved cell vitality and insulin secretion of INS-1 cells induced by high glucose. We found Nifu significantly inhibited pro-oxidative factors (ROS, MDA) and promoted anti-oxidative factors (SOD, GSH-PX, CAT). Meanwhile, qRT-PCR and Western blot results showed that inflammatory and apoptosis factors were remarkably inhibited by Nifu. Further research indicated that Nifu clearly suppressed the activation of the STAT3/SOCS3 signaling pathway. In conclusion, Nifu can significantly improve the insulin secretion function, protect oxidative stress injury, and reduce inflammatory response and apoptosis in high glucose-induced INS-1 cells. Therefore, Nifu has a new positive effect on maintaining the normal function of pancreatic islet cells in a high glucose environment and provides new drug candidates for the treatment and prevention of diabetes.

Adenoid cystic carcinoma of the gastroesophageal junction: A case report.

RATIONALE: Adenoid cystic carcinoma (ACC) rarely occurs in the digestive tract, particularly in the gastroesophageal junction. PATIENT CONCERNS: A 44-year-old male vomiting blood was admitted to our hospital. Endoscopic ultrasound showed a 2.2 × 3.0 cm submucosal tumor in the gastroesophageal junction. DIAGNOSIS: According to the histopathological examination, the tumor was composed predominantly of ductal epithelial and myoepithelial cells. Immunohistochemical staining revealed that the tumor expressed cytokeratin, cluster of differentiation 117, p63, and calponin. Based on these findings, ACC was diagnosed. INTERVENTIONS: Endoscopic submucosal dissection (ESD) was performed to remove the tumor. As the margins of the ESD specimen were positive, the patient underwent total gastrectomy with D2 lymphadenectomy. Finally, neither residual tumor nor lymphatic metastasis was detected in the surgical specimens. OUTCOMES: No sign of recurrence has been detected during 36 months of follow-up as of October 2018. LESSONS: ESD may be an alternative treatment for cardial ACC invading the submucosa.

Long-term adjuvant imatinib treatment for a patient who underwent complete resection of a localized recurrent gastrointestinal stromal tumor after preoperative imatinib treatment: A case report.

RATIONALE: The efficiency and tolerance of long-term adjuvant imatinib treatment for patient who underwent complete resection of a localized recurrent gastrointestinal stromal tumor (GIST) was unknown. PATIENT CONCERNS: A 45-year-old man underwent complete resection of an intestinal GIST in August 2001. Four years later, a giant (11 × 8 × 6 cm) recurrent GIST located in the retroperitoneum was detected. DIAGNOSIS: The recurrent tumor was positive for CD117 by immunohistochemistry. INTERVENTIONS: The recurrent tumor was completely resected after 4 months of effective imatinib treatment (400 mg/day), and the patient continued imatinib treatment postoperatively. In June 2011, imatinib treatment was stopped for 3 weeks because of hepatitis B infection, and resumed with a reduced dose level of 300 mg/day when liver function recovered. In March 2017, imatinib treatment was interrupted again for 12 days because the patient underwent cholecystectomy. OUTCOMES: In December 2017, a computed tomography scan showed no signs of tumor recurrence. To date, the patient has been under adjuvant imatinib treatment for >12 years without severe side effects. The plasma concentration of imatinib (detected in February 2018) was trough concentration (Cmin) 1015.7 ng/mL and peak concentration (Cmax) 1550.5 ng/mL. LESSONS: This case report highlights the active role of long-term (>12 years) imatinib treatment after complete resection of localized recurrent GIST.

Von Willebrand factor and coagulation factor VIII in Moyamoya disease associated with Graves' disease: A case report.

The present study reported the case of a Chinese boy who was diagnosed with Moyamoya disease (MMD) associated with Graves' disease (GD). An overactivation of von Willebrand factor (vWF) and coagulation factor VIII (FVIII) was identified in the plasma of the patient. Thiamazole and metoprolol treatment was thus administrated. After 2 months of treatment, the patient's thyroid function returned to normal and the neurological symptoms improved gradually. At the same time, the activities of vWF and FVIII were depressed. During the 20-month follow-up, information regarding the neurological symptoms, cerebrovascular imaging, thyroid function, thyroid autoantibodies and coagulation parameters was collected. High levels of thyroid autoantibodies persisted throughout the follow-up period, while other coagulation parameters remained in the normal range. In conclusion, considering the vital role of vWF and FVIII in vascular diseases, it is hypothesized that these two factors may serve an important role in the occurrence of GD associated with MMD.

Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

[Noncoding RNA in the Regulation of Hepatic Glucose and Lipid Metabolism].

In the past decades, diabetes, in particular type 2 diabetes (T2D)mainly characterized by global insulin resistance and pancreatic beta cell failure, had become epidemic and a severe public health threat worldwide with the development of economy and change of lifestyle.The interactions between environment factors and genetic background play vital roles in the development and progression of T2D.More recently, it had been revealed that non-coding RNA including microRNA (miRNA)and long noncoding RNA (LncRNA)are widely involved inthe regulation of glucose and lipid metabolism. So far, it had been established that deregulated miRNA and LncRNA profile in main metabolic tissues is tightly associated with T2D,and intensive studies on non-coding RNAs had shed light on understanding the pathogen-esis of T2D.The current review aimed to briefly summarize and discuss the latest findings regarding the role and mechanism of miRNAs and LncRNAs in the regulation hepatic glucose and lipid metabolism.

Risk factor detection for heart disease by applying text analytics in electronic medical records.

In the United States, about 600,000 people die of heart disease every year. The annual cost of care services, medications, and lost productivity reportedly exceeds 108.9 billion dollars. Effective disease risk assessment is critical to prevention, care, and treatment planning. Recent advancements in text analytics have opened up new possibilities of using the rich information in electronic medical records (EMRs) to identify relevant risk factors. The 2014 i2b2/UTHealth Challenge brought together researchers and practitioners of clinical natural language processing (NLP) to tackle the identification of heart disease risk factors reported in EMRs. We participated in this track and developed an NLP system by leveraging existing tools and resources, both public and proprietary. Our system was a hybrid of several machine-learning and rule-based components. The system achieved an overall F1 score of 0.9185, with a recall of 0.9409 and a precision of 0.8972.

Primary gastrointestinal stromal tumor of the liver treated with sequential therapy.

A 67-year-old female presented with a primary hepatic gastrointestinal stromal tumor that was detected by computed tomography and diagnosed based on histopathological and genetic analyses. The tumor was microscopically composed of spindle cells and epithelioid cells, and immunohistochemistry results showed positive staining for CD117 and CD34 expression. A genetic analysis revealed a heterozygous point mutation and deletion in exon 11 of c-KIT. After an R0 resection, imatinib mesylate was administered for 1 year until its use was discontinued due to severe side effects. Two years after the original operation, the tumor recurred in the residual liver and was completely resected again. Imatinib mesylate was administered for 2 years until it was replaced by sunitinib malate because of disease progression. The patient has survived for 53 mo after undergoing a sequential therapy consisting of surgical excision, imatinib and sunitinib.

Bowen's disease with features resembling myrmecia wart.

We report the clinical and pathological findings of two cases of Bowen's disease (BD) with features resembling myrmecia wart, and tried to find evidence of human papillomavirus (HPV) infection in such lesions by immunohistological staining, genotyping systems, polymerase chain reaction (PCR) and electron microscopy. Both cases manifested unique barnacle-like hyperkeratotic nodules or plaques clinically, and microscopically proliferation of atypical keratinocytes involving the entire thickness of the epidermis, hypergranulosis with eosinophilic and/or basophilic inclusion bodies, features that mimicked myrmecia wart. Electron microscopy revealed myrmecia inclusion-like large intranuclear and cytoplasmic electron-dense bodies. Immunohistological staining with anti-HPV antibody, genotyping systems for HPV infection and specific PCR designed to detect HPV-1 L1 sequences failed to detect evidence of HPV infection. P16(INK4a) was overexpressed in the atypical keratinocytes of both cases. This finding suggests that the pathogenesis of these two BD may involve certain unknown or undetectable HPV, or reflect disturbances of the Rb signaling pathway unrelated to HPV infection. The unique "myrmecioid" clinicopathological features in our cases suggest that this type of lesion may be a new variant of BD.