High-Intensity Focused Ultrasound Decreases Subcutaneous Fat Tissue Thickness by Increasing Apoptosis and Autophagy.

High-intensity focused ultrasound (HIFU) leads to decreased subcutaneous adipose tissue (SAT) thickness via heat-induced adipocyte necrosis. Heat can induce adipocyte apoptosis and autophagy, and it is known that nuclear or mitochondrial p53 is involved in apoptosis and autophagy. However, whether HIFU leads to apoptosis or autophagy is unclear. We evaluated whether HIFU decreases SAT thickness via p53-related apoptosis or autophagy in high-fat diet (HFD)-fed animals. The expression of nuclear and mitochondrial p53 was increased by HIFU. HIFU also led to decreased expression of BCL2/BCL-xL (an antiapoptotic signal), increased expression of BAX/BAK (an apoptotic signal), increased levels of cleaved caspase 3/9, and increased numbers of apoptotic cells as evaluated by TUNEL assay. Furthermore, HIFU led to increased levels of ATG5, BECN1, and LC3II/LC3I, and decreased levels of p62, a marker of increased autophagy. The thickness of SAT was decreased by HIFU. In conclusion, HIFU led to nuclear and mitochondrial p53 expression, which led to apoptosis and autophagy, and eventually decreased SAT thickness in HFD-fed animals.

COVID-19 Sequelae and Their Implications on Social Services.

BACKGROUND: The impact of persistent coronavirus disease 2019 (COVID-19) symptoms on quality of life remains unclear. This study aimed to describe such persistent symptoms and their relationships with quality of life, including clinical frailty and subjective health status. METHODS: A prospective longitudinal 3-month follow-up survey monitored symptoms, health quality, support needs, frailty, and employment. RESULTS: A total of 82 patients with a mean age of 52 years (ranging from 23-84 years) were enrolled, including 48 (58.6%) men, and 34 (41.5%) women. The fully active status decreased from 87.8% before admission to 78.1% post discharge. Two patients (2.4%) were ambulatory and capable of all self-care but unable to carry out any work-related activities 12 weeks after discharge. Clinical frailty scale (CFS) levels 1, 2, 3 and 4 changed drastically between admission and 12 weeks later after discharge. Just after admission, the median EuroQol visual analogue scales (EQ-VAS) was 82.23 (± 14.38), and it decreased to 78.10 (± 16.02) 12 weeks after discharge; 62 (75.6%) of patients reported at least one symptom 12 weeks after discharge. The most frequent symptom was fatigue followed by smell disorder, anxiety, sleep disorder, headache, depressive mood, dyspnea, and taste disorder. CFS was definitively associated with fatigue. Decreased EQ-VAS was associated with fatigue and palpitation, cough, taste disorder, and chest pain. EQ-VAS was worse in women (28%) than in men. Compared with regular outpatient clinic visits before admission, 21 patients (25.6%) reported increased outpatient clinic visits, one (1.4%) reported readmission, and one (1.4%) reported emergency room visits. Six of the 54 (77.1%) patients who were employed before admission lost their jobs. And most vulnerable type was self-employed, because three self-employed job workers were not working at 12 weeks after discharge. CONCLUSION: COVID-19 sequelae should not be underestimated. We find a decrease in health quality and increase in psychological problems in discharged COVID-19 patients, and some patients experience unemployment. The number of patients suffering from COVID-19 sequelae would not be negligible considering there are more than one million COVID-19 infection cases in Korea. Hence, the government should start a systematic monitoring system for discharged patients and prepare timely medical and social interventions accordingly.

Older Workers with Physically Demanding Jobs and their Cognitive Functioning.

Although employment can provide older people with both financial and nonfinancial rewards, it is questionable whether those benefits extend to all older workers, particularly those with physically demanding jobs. This study aimed to examine whether the perceived level of physical demands placed on older workers 55 or older is significantly associated with their cognitive function. Using the Health and Retirement Study (HRS) 2010 wave, we analyzed two domains of cognition: verbal episodic memory and reasoning. After controlling for demographics and risk factors for age-related cognitive deterioration, the perceived level of physical demands placed on older workers was still significantly and negatively linked with both memory and reasoning domains of cognition. Older workers with more physically demanding jobs tended to have poorer cognitive function. Further longitudinal studies are needed to confirm this relationship. Supplementary Information: The online version contains supplementary material available at 10.1007/s12126-020-09404-8.

Attenuation Effect of Radiofrequency Irradiation on UV-B-Induced Skin Pigmentation by Decreasing Melanin Synthesis and through Upregulation of Heat Shock Protein 70.

Excess melanin deposition in the skin causes cosmetic problems. HSP70 upregulation decreases microphthalmia-associated transcription factor (MITF) expression, which eventually decreases tyrosinase activity and melanogenesis. Ultraviolet (UV) radiation upregulates p53, which increases the melanocortin receptor (MC1R) and MITF. Furthermore, HSP70 decreases p53 and radiofrequency irradiation (RF) increases HSP70. We evaluated whether RF increased HSP70 and decreased p53, consequently decreasing the MITF/tyrosinase pathway and melanogenesis in UV-B radiated animal skin. Various RF combinations with 50, 100, and 150 ms and 5, 10, and 15 W were performed on the UV-B radiated mouse skin every 2 d for 28 d. When RF was performed with 100 ms/10 W, melanin deposition, evaluated by Fontana-Masson staining, decreased without skin crust formation in the UV-B radiated skin. Thus, we evaluated the effect of RF on decreasing melanogenesis in the HEMn and UV-B radiated skin at a setting of 100 ms/10 W. HSP70 expression was decreased in the UV-B radiated skin but was increased by RF. The expression of p53, MC1R, and MITF increased in the UV-B radiated skin but was decreased by RF. The expression of p53, MC1R, and MITF increased in the α-MSH treated HEMn but was decreased by RF. The decreasing effects of RF on p53, MC1R, CREB and MITF were higher than those of HSP70-overexpressed HEMn. The decreasing effect of RF on p53, MC1R, CREB, and MITF disappeared in the HSP70-silenced HEMn. MC1R, CREB, and MITF were not significantly decreased by the p53 inhibitor in α-MSH treated HEMn. RF induced a greater decrease in MC1R, CREB, and MITF than the p53 inhibitor. Therefore, RF may have decreased melanin synthesis by increasing HSP70 and decreasing p53, thus decreasing MC1R/CREB/MITF and tyrosinase activity.

Standardization of the Manufacturing Process of Bee Venom Pharmacopuncture Containing Melittin as the Active Ingredient.

BACKGROUND: Pharmacopuncture is a unique treatment in oriental medicine that combines chemical stimulation with conventional acupuncture. However, there are no standardized methods for preparing the herbal medicines used in pharmacopuncture, and it is not clear whether the active ingredients are safe and stable. Several studies have investigated nonstandardized preparation processes, but few investigations have addressed safety and preparation methods. Pharmacopuncture may provide an alternative treatment for incurable diseases. However, it must be as valid and safe as standardized medicine. In this way, the present project may contribute to the industrialization of medicine in Korea. It may also expand health insurance coverage by promoting evidence-based medical insurance benefits. Thus, the present study attempted to standardize and improve the raw materials, preparation, and efficacy of bee venom pharmacopuncture (BVP), which is a highly effective technique in oriental medicine. METHOD: To purify the crude bee venom, the extract was subjected to a stepped-gradient open column (ODS-A; 120 Å, 150 mesh). Using this method, the yield of melittin was significantly increased and the allergen proteins were effectively removed. The melittin content of the purified bee venom was determined using HPLC, and the product was then diluted to 0.1 mg/mL using injection water in preparation for BVP. RESULTS: In the present study, we standardized the purification process to provide safe and stable BVP by increasing the main effective components and eliminating allergens. This study will be seminal in the industrialization and regulation of BVP. CONCLUSION: We developed an effective strategy for melittin purification and allergen removal from bee venom to create safe BVP.

Safety of essential bee venom pharmacopuncture as assessed in a randomized controlled double-blind trial.

ETHNOPHARMACOLOGICAL RELEVANCE: While bee venom (BV) pharmacopuncture use is common in Asia, frequent occurrence of allergic reactions during the treatment process is burdensome for both practitioner and patient. AIM OF THE STUDY: This study compared efficacy and safety in isolated and purified essential BV (eBV) pharmacopuncture filtered for phospholipase A2 (PLA2) and histamine sections, and original BV to the aim of promoting safe BV pharmacopuncture use. MATERIALS AND METHODS: In in vitro, we examined the effect of BV and eBV on nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages, and clinically, 20 healthy adults aged 20-40 years were randomly allocated and administered eBV 0.2mL and BV pharmacopuncture 0.2mL on left and right forearm, respectively, and physician, participant, and outcome assessor were blinded to treatment allocation. Local pain, swelling, itching, redness, wheals, and adverse reactions were recorded by timepoint. RESULTS: eBV and BV exhibited similar inhibitory effects on NO production. Also, in comparison between eBV and BV pharmacopuncture administration areas on each forearm, eBV displayed significantly lower local pain at 24h post-administration (P=0.0062), and less swelling at 30min (P=0.0198), 2 (P=0.0028), 24 (P=0.0068), and 48h post-administration (P=0.0253). eBV also showed significantly less itching at 24 (P=0.0119), 48 (P=0.0082), and 96h (P=0.0141), while redness was significantly less at 30min (P=0.0090), 6 (P=0.0005), and 24h (P<0.0001). Time-by-treatment interactions were statistically significant for itching and redness (P<0.001, and P<0.001, respectively), and all original BV pharmacopuncture administered regions showed a tendency toward more severe itching and redness in later measurements. CONCLUSIONS: eBV and BV displayed comparable anti-inflammatory effects, and eBV pharmacopuncture presented less local allergic reactions.

Longitudinal Relationships Between Productive Activities and Functional Health in Later Years: A Multivariate Latent Growth Curve Modeling Approach.

This study examined the longitudinal relationships between functional health in later years and three types of productive activities: volunteering, full-time, and part-time work. Using the data from five waves (2000-2008) of the Health and Retirement Study, we applied multivariate latent growth curve modeling to examine the longitudinal relationships among individuals 50 or over. Functional health was measured by limitations in activities of daily living. Individuals who volunteered, worked either full time or part time exhibited a slower decline in functional health than nonparticipants. Significant associations were also found between initial functional health and longitudinal changes in productive activity participation. This study provides additional support for the benefits of productive activities later in life; engagement in volunteering and employment are indeed associated with better functional health in middle and old age.

Prevalence of Porphyromonas gingivalis fimA genotypes in the peri-implant sulcus of Koreans assessed using a new primer.

PURPOSE: Porphyromonas gingivalis fimA is a virulence factor associated with periodontal diseases, but its role in the pathogenesis of peri-implantitis remains unclear. We aimed to evaluate the relationship between the condition of peri-implant tissue and the distribution of P. gingivalis fimA genotypes in Koreans using a new primer. METHODS: A total of 248 plaque samples were taken from the peri-implant sulci of 184 subjects. The control group consisted of sound implants with a peri-implant probing depth (PD) of 5 mm or less with no bleeding on probing (BOP). Test group I consisted of implants with a peri-implant PD of 5 mm or less and BOP, and test group II consisted of implants with a peri-implant PD of more than 5 mm and BOP. DNA was extracted from each sample and analyzed a using a polymerase chain reaction (PCR) with P. gingivalis-specific primers, followed by an additional PCR assay to differentiate the fimA genotypes in P. gingivalis-positive subjects. RESULTS: The Prevalence of P. gingivalis in each group did not significantly differ (P>0.05). The most predominant fimA genotype in all groups was type II. The prevalence of type Ib fimA was significantly greater in test group II than in the control group (P<0.05). CONCLUSIONS: The fimA type Ib genotype of P. gingivalis was found to play a critical role in the destruction of peri-implant tissue, suggesting that it may be a distinct risk factor for peri-implantitis.

Directed evolution of human heavy chain variable domain (VH) using in vivo protein fitness filter.

Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.

Survival of 352 titanium implants placed in 181 patients: a 4-year multicenter field study.

PURPOSE: The aim of this retrospective chart review was to evaluate the four-year survival rate of a titanium implant system. METHODS: A total of 352 sand-blasted, thermally acid-etched titanium implants were inserted into 181 partially or completely edentulous patients. Their cumulative survival rate was evaluated retrospectively. Associated factors, such as the implant distribution and treatment type were included in the evaluation. RESULTS: The implants were equally distributed between the maxilla (52.3%) and the mandible (47.7%). 48 implants (13.6%) were placed in the anterior region and 304 implants (86.4%) in the posterior region. The majority of the implants were inserted into bone of type II and III quality (89.8%) and volume (quantity B and C, 87.2%). Most of the implants (70.7%) were restored as single crowns; 28.7% supported a bridge construction and 0.6% a full denture. Only one implant failed, resulting in a four-year cumulative survival rate of 99.7%. CONCLUSIONS: The implant system showed an excellent four-year survival rate. It proved to be a safe and predictable means for restoration of the dentition in partially or completely edentulous patients.

Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation.

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.

Purification and characterization of a recombinant anti-angiogenic kringle fragment expressed in Escherichia coli: Purification and characterization of a tri-kringle fragment from human apolipoprotein (a) (kringle IV (9)-kringle IV (10)-kringle V).

A kringle fragment (type IV (9)-IV (10)-V) from human apolipoprotein (a) (called LK68) was expressed in an inclusion body in Escherichia coli. The LK68 in this inclusion body was rendered soluble with urea, and efficiently refolded via oxidation in the presence of re-dox couple. The refolded LK68 was then purified via two steps of ion exchange chromatography, concentrated via preparative reversed-phase chromatography, and freeze-dried, at a final yield of approximately 30%. The purified LK68 exhibited profound affinity for lysine and fibrinogen, which suggests the proper folding of the kringle fragment, and also indicates that the native characteristics of apolipoprotein (a) were preserved. The purified LK68 was determined to be highly homogeneous upon reversed-phase HPLC analysis and size-exclusion HPLC analysis, in the presence of 20% (v/v) acetonitrile. However, on size-exclusion HPLC analysis without acetonitrile, it was determined to be somewhat heterogeneous, and this was corroborated by native analyses, including native PAGE and IEF.

Production of the kringle fragments of human apolipoprotein(a) by continuous lactose induction strategy.

A novel lactose induction strategy for the production of rhLK68, the kringle fragments of human apolipoprotein(a) (apo(a)) as a novel anti-angiogenic protein, was investigated. A scale-up of the production was accompanied by a decrease in expression level, and severe aggregation occurred during the solubilization of rhLK68 from the inclusion body during a conventional single introduction of lactose. To overcome this problem, a continuous induction strategy was applied where lactose was mixed with glycerol and fed continuously in a dissolved oxygen (DO)-stat manner. With the sub-optimal feed medium consisted of 1:50 of lactose/glycerol (w/w), the expression level reached 16% of the total cellular protein, which was 1.6-fold higher than that obtained from the conventional lactose induction. Moreover, the solubilization yield of rhLK68 from the inclusion body increased from 30 +/- 5 to 85 +/- 3% compared to the conventional single introduction of lactose. This result suggests that the continuous lactose induction strategy beneficially influenced the expression level of rhLK68 and the quality of its inclusion body.