New Implications of Metabolites and Free Fatty Acids in Quality Control of Crossbred Wagyu Beef during Wet Aging Cold Storage.

Efficient cold-chain delivery is essential for maintaining a sustainable global food supply. This study used metabolomic analysis to examine meat quality changes during the "wet aging" of crossbred Wagyu beef during cold storage. The longissimus thoracic (Loin) and adductor muscles (Round) of hybrid Wagyu beef, a cross between the Japanese Black and Holstein-Friesian breeds, were packaged in vacuum film and refrigerated for up to 40 days. Sensory evaluation indicated an increase in the umami and kokumi taste owing to wet aging. Comprehensive analysis using gas chromatography-mass spectrometry identified metabolite changes during wet aging. In the Loin, 94 metabolites increased, and 24 decreased; in the Round, 91 increased and 18 decreased. Metabolites contributing to the umami taste of the meat showed different profiles during wet aging. Glutamic acid increased in a cold storage-dependent manner, whereas creatinine and inosinic acid degraded rapidly even during cold storage. In terms of lipids, wet aging led to an increase in free fatty acids. In particular, linoleic acid, a polyunsaturated fatty acid, increased significantly among the free fatty acids. These results provide new insight into the effects of wet aging on Wagyu-type beef, emphasizing the role of free amino acids, organic acids, and free fatty acids generated during cold storage.

Autotaxin is a novel surrogate marker for oxaliplatin-related sinusoidal obstruction syndrome in patients with colorectal liver metastasis.

BACKGROUND: Chemotherapy for colorectal liver metastasis (CRLM) has improved dramatically over the past few decades. However, sinusoidal obstruction syndrome (SOS) induced by oxaliplatin leads to increased severe morbidity after hepatectomy for CRLM. Autotaxin is a novel liver fibrosis marker known to be taken up and metabolized by sinusoidal endothelial cells. This study aimed to evaluate whether autotaxin levels could be a novel surrogate marker of SOS for CRLM. METHODS: We retrospectively evaluated 73 consecutive patients who underwent hepatectomy for CRLM, and assessed the relationship between their preoperative autotaxin levels and SOS. RESULTS: Median autotaxin level was 0.750 mg/L. Preoperative oxaliplatin-based chemotherapy for CRLM was administered to 51 patients, and SOS was histologically observed in 45 patients. Patients who received the oxaliplatin-based chemotherapy had significantly higher autotaxin levels than those who did not (p = 0.038). Furthermore, autotaxin levels were higher in patients with SOS than in those without (p = 0.011). Univariate and multivariate analyses revealed that autotaxin level can be an independent predictive factor for SOS preoperatively (p = 0.001). CONCLUSIONS: Autotaxin level is a noninvasive and promising surrogate marker for predicting SOS before surgical resection for CRLM.

Two cases of follicular lymphoma with MYC gene abnormalities that presented with bone marrow necrosis.

Bone marrow necrosis (BMN) occurs most frequently in hematological malignancies and sometimes in non-hematological disorders. Lymphoid diseases causing necrosis are regarded as high-grade disease. B-lymphoblastic leukemia/lymphoma is the most common malignant cause of BMN. Here, we present two patients with follicular lymphoma (FL) and MYC gene abnormalities who developed BMN. In one case of BMN, the necrosis disappeared in response to chemotherapy, and the patient survived with complete remission. In the other case, BMN remained even after chemotherapy, and effective chemotherapy could not be administered due to suppressed hematopoiesis, which led to the lymphoma worsening and the patient's death. Indolent lymphomas, such as FL, as in these cases, have the potential to develop BMN. It is important to detect the development of BMN and administer chemotherapy early to improve patient prognosis, since severe BMN prevents patients from receiving effective treatment.

Molecular and expressional characterization of tumor heterogeneity in pulmonary carcinosarcoma.

The genetic concordance and heterogeneity of the two components of pulmonary carcinosarcoma (PCS), carcinoma, and sarcoma, have not been fully elucidated because of its rare occurrence. We performed targeted sequencing of the carcinoma and sarcoma components of four PCSs to identify genetic similarities and differences. Formalin-fixed paraffin-embedded tissue samples were macroscopically or microscopically dissected. DNA was extracted from each component, and genetic alterations were analyzed separately. Moreover, we performed RNA-seq analysis on both components of one PCS to compare differences in gene expression profiles. The carcinoma part consisted of adenocarcinoma in two cases, squamous cell carcinoma in one, and adenosquamous carcinoma in the last. TP53 mutation was observed in three samples from the trunk, although it was detected only in the sarcoma part in one case. No specific driver gene mutation was observed; however, KRAS mutations were observed in one case in the trunk. RNA-seq analysis revealed that the rhabdomyosarcoma component expressed various genes related to muscle development, whereas the carcinoma component did not; and that gene expression overall was completely different between the two components. Our study revealed that the two different components of PCS shared common gene mutations in most cases. Although gene expression was different among components, if driver genes such as KRAS were detected in PCS, molecular targeted therapy could be beneficial even when the tumor contains a sarcoma component.

[Development of a risk prediction model and a sample risk chart for long-term care certification based on the functional health of older adults].

Objectives　The first aim of this study was to develop risk prediction models based on age, sex, and functional health to estimate the absolute risk of the 3-year incidence of long-term care certification and to evaluate its performance. The second aim was to produce risk charts showing the probability of the incident long-term care certification as a tool for prompting older adults to engage in healthy behaviors.Methods　This study's data was obtained from older adults, aged ≥65 years, without any disability (i.e., they did not certify≥care level 1) and residing in Yabu, Hyogo Prefecture, Japan (n=5,964). A risk prediction model was developed using a logistic regression model that incorporated age and the Kihon Checklist (KCL) score or the Kaigo-Yobo Checklist (KYCL) score for each sex. The 3-year absolute risk of incidence of the long-term care certification (here defined as≥care level 1) was then calculated. We evaluated the model's discrimination and calibration abilities using the area under the receiver operating characteristic curves (AUC) and the Hosmer-Lemeshow goodness-of-fit test, respectively. For internal validity, the mean AUC was calculated using a 5-fold cross-validation method.Results　After excluding participants with missing KCL (n=4) or KYCL (n=1,516) data, we included 5,960 for the KCL analysis and 4,448 for the KYCL analysis. We identified incident long-term care certification for men and women during the follow-up period: 207 (8.2%) and 390 (11.3%) for KCL analysis and 128 (6.6%) and 256 (10.2%) for KYCL analysis, respectively. For calibration, the χ2 statistic for the risk prediction model using KCL and KYCL was: P=0.26 and P=0.44 in men and P=0.75 and P=0.20 in women, respectively. The AUC (mean AUC) in the KCL model was 0.86 (0.86) in men and 0.83 (0.83) in women. In the KYCL model, the AUC was 0.86 (0.85) in men and 0.85 (0.85) in women. The risk charts had six different colors, suggesting the predicted probability of incident long-term care certification.Conclusions　The risk prediction model demonstrated good discrimination, calibration, and internal validity. The risk charts proposed in our study are easy to use and may help older adults in recognizing their disability risk. These charts may also support health promotion activities by facilitating the assessment and modification of the daily behaviors of older adults in community settings. Further studies with larger sample size and external validity verification are needed to promote the widespread use of risk charts.

An Autopsy Case of Multicentric Castleman Disease Presenting with Severe Jaundice.

A 70-year-old man with multicentric Castleman disease (MCD) was admitted to our hospital with jaundice and ascites. Elevations in his bilirubin and interleukin-6 levels were noted, and computed tomography revealed hepatic atrophy and portal vein and bile duct disorders. Steroid therapy was started for MCD, but he died of hepatic failure. An autopsy revealed that the MCD activity was mild, but advanced fibrosis and cholestasis were observed in the liver. Mild infiltration of interleukin-6-positive plasma cells was noted in the highly fibrotic area of the liver. Although rare, liver and biliary tract damage may be also considered organ disorders of MCD.

A mucinous cystic neoplasm of the pancreas containing an undifferentiated carcinoma component and harboring the NRAS driver mutation.

A woman in her 20s visited our hospital with a chief complaint of abdominal distension and back pain. She was diagnosed with a cystic tumor (diameter 16 cm) in the tail of the pancreas and underwent a combined distal pancreatectomy, splenectomy, and left adrenalectomy. Histopathologically, the tumor presented as a mucinous cystic neoplasm with an undifferentiated carcinoma component of the pancreas. In addition, the cells demonstrated a partial rhabdoid-like morphology. These findings were considered relatively typical for a mucinous cystic neoplasm in the tail of the pancreas in a young woman. However, NRAS mutation, which is rare in pancreatic tumors, was detected.

Solid-phase synthesis and bioactivity evaluation of cherimolacyclopeptide E.

Cherimolacylopeptide E (1) is a cyclic hexapeptide isolated from the seeds of Annona cherimola. Peptide 1 reportedly exhibits potent cytotoxicity against KB cells (IC50 0.017 µM). To confirm the structure and bioactivity of 1, we conducted a total synthesis of its proposed structure. The synthesis was accomplished via solid-phase peptide elongation and macrocyclization by employing Fmoc/OAll-protected amino acids on 2-Cl-trityl resin. NMR analysis revealed that synthetic 1 exists in two conformations in pyridine-d5 . As the spectroscopic data of the major conformer of synthetic 1 were consistent with those of natural 1, the structure of cherimolacyclopeptide E was confirmed to be 1. However, our synthetic 1 exhibited low cytotoxicity against KB cells (IC50 > 100 µM). In contrast to previously-reported findings, our synthetic 1 exhibited little antibacterial activity against Escherichia coli.

Simultaneous Quantification of BCR-ABL and Bruton Tyrosine Kinase Inhibitors in Dried Plasma Spots and Its Application to Clinical Sample Analysis.

BACKGROUND: Recent reports highlight the importance of therapeutic drug monitoring (TDM) of BCR-ABL and Bruton tyrosine kinase inhibitors (TKIs); thus, large-scale studies are needed to determine the target concentrations of these drugs. TDM using dried plasma spots (DPS) instead of conventional plasma samples is a promising approach. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of BCR-ABL and Bruton TKIs for further TDM studies. METHODS: A 20-µL aliquot of plasma was spotted onto a filter paper and dried completely. Analytes were extracted from 2 DPS using 250 µL of solvent. After cleanup by supported liquid extraction, the sample was analyzed by LC-MS/MS. Applicability of the method was examined using samples of patients' DPS transported by regular mail as a proof-of-concept study. The constant bias and proportional error between plasma and DPS concentrations were assessed by Passing-Bablok regression analysis, and systematic errors were evaluated by Bland-Altman analysis. RESULTS: The method was successfully validated over the following calibration ranges: 1-200 ng/mL for dasatinib and ponatinib, 2-400 ng/mL for ibrutinib, 5-1000 ng/mL for bosutinib, and 20-4000 ng/mL for imatinib and nilotinib. TKI concentrations were successfully determined for 93 of 96 DPS from clinical samples. No constant bias between plasma and DPS concentrations was observed for bosutinib, dasatinib, nilotinib, and ponatinib, whereas there were proportional errors between the plasma and DPS concentrations of nilotinib and ponatinib. Bland-Altman plots revealed that significant systematic errors existed between both methods for bosutinib, nilotinib, and ponatinib. CONCLUSIONS: An LC-MS/MS method for the simultaneous quantification of 6 TKIs in DPS was developed and validated. Further large-scale studies should be conducted to assess the consistency of concentration measurements obtained from plasma and DPS.

Healthy lifestyle behaviors and transitions in frailty status among independent community-dwelling older adults: The Yabu cohort study.

Frailty is a dynamic condition that can worsen or improve. Although previous studies have identified demographic and intrinsic factors associated with transitions in frailty status, less evidence is available regarding associations with daily activities. This longitudinal study examined associations of behavioral factors with incident frailty, adverse events (e.g. residence in a nursing home, hospitalization, and death), and frailty improvement among older adults. The analysis included data from 3,769 independent older adults aged 65 years or more (mean age, 76.3 ± 7.2years; proportion of frail participants, 30.1%) from the Yabu cohort study (baseline, 2012; follow-up, 2017). The Kaigo-Yobo Checklist, a standardized questionnaire, was used to assess frailty status (non-frail or frail) and seven behavioral factors: farming, shopping, exercise, eating habit, intellectual activity, social participation, and smoking. At the 5-year follow-up survey, the proportions of participants transitioning from non-frail to frail, from non-frail to incident adverse events, from frail to non-frail, and from frail to incident adverse events were 16.8%, 13.9%, 15.2%, and 50.1%, respectively. In the analysis of non-frail participants, farming, exercise, intellectual activity, and social participation were associated with lower odds of becoming frail and experiencing adverse events. In the analysis for frail participants, farming, intellectual activity, and social participation were significantly associated with improvement in frailty status, and farming, shopping, and social participation were associated with lower odds of incident adverse events. These findings suggest that farming, healthy daily activities, and social participation help improve and prevent frailty/adverse events.

The Synthesis of YNU-5 Zeolite and Its Application to the Catalysis in the Dimethyl Ether-to-Olefin Reaction.

During prior investigations of the synthesis of the novel zeolite YNU-5 (YFI), it was found that a very slight amount of an impurity phase contaminated the desired zeolitic phase. This impurity was very often ZSM-5 (MFI). The phase composition was determined to be sensitive to the water in the synthesis mixture, and it was possible to obtain a pure phase and also to intentionally generate a specific impurity phase. In the present work, trials based on the dimethyl ether-to-olefin (DTO) reaction using a fixed-bed downflow reactor were performed to assess the effect of the purity of YNU-5 on its catalytic performance. Dealuminated pure YNU-5 exhibited rapid deactivation due to coking at time on stream (TOS) values exceeding 5 min. Surprisingly, this deactivation was greatly suppressed when the material contained a trace amount of ZSM-5 consisting of nano-sized particles. The formation of ZSM-5 nanoparticles evidently improved the performance of the catalytic system during the DTO reaction. The product distributions obtained from this reaction using highly dealuminated and very pure YNU-5 resembled those generated by 12-ring rather than 8-ring zeolite catalysts. The high selectivity for desirable C3 and C4 olefins during the DTO reaction over YNU-5 is beneficial.

Secondary INI1-deficient rhabdoid tumors of the central nervous system: analysis of four cases and literature review.

Atypical teratoid/rhabdoid tumors (AT/RT) are rare, highly malignant neoplasms of the central nervous system that predominantly occur in infants, and are characterized by the presence of rhabdoid cells and inactivation of INI1 or (extremely rarely) BRG1. The vast majority of AT/RT are recognized as primary tumors; however, rare AT/RT or INI1-deficient RT arising from other primary tumors have been reported. To better characterize secondary RT, we performed a histological and molecular analysis of four RT arising from pleomorphic xanthoastrocytoma (PXA), anaplastic PXA, low-grade astrocytoma, or ependymoma. Histologically, although conventional AT/RT are usually not largely composed of rhabdoid cells, three secondary RT were composed mainly of rhabdoid cells, two of which arising from (anaplastic) PXA exhibited marked nuclear pleomorphism reminiscent of that in the precursor lesions. Regarding INI1 alterations, although mutations including small indels are frequent in conventional AT/RT, only in one secondary RT had a mutation. Moreover, together with previously reported cases, biallelic INI1 inactivation in secondary RT was mostly due to biallelic focal and/or broad deletions. Although conventional AT/RT have stable chromosomal profiles, i.e., the frequency of copy number changes involving chromosomes other than chromosome 22 is remarkably low, our array comparative genomic hybridization analysis revealed numerous copy number changes in the secondary RT. In conclusion, secondary RT of the central nervous system are clinicopathologically and molecularly different from conventional pediatric AT/RT, and a nosological issue is whether these secondary RT should be called secondary "AT/RT" as most of the reported cases were.

[Process and outcome evaluation of community-based strategies for frailty prevention in the elderly with the help of the senior workforce at Silver Human Resources Centers in Yabu City, Japan].

Objectives　Preventing frailty is a crucial issue in aging societies such as Japan. In 2011, we launched an action research project in Yabu City, Hyogo Prefecture, to develop effective community-based strategies to prevent frailty in the elderly. We attempted to introduce community-based frailty prevention classes in every administrative district with the help of the senior workforce at Silver Human Resources Centers. This study aimed to evaluate the effectiveness and the applicability to different communities of this strategy, which will be called the "Yabu model."Methods　Using PAIREM (Plan, Adoption, Implementation, Reach, Effectiveness, Maintenance) framework, we evaluated the effectiveness and the applicability to different communities of the Yabu model. To evaluate its effectiveness, we conducted a baseline and follow-up survey of residents aged 65 years or older in 2012 (n=7,287, 90.7% response rate) and 2017 (n=8,157, 85.7%), using a mailed self-administered questionnaire.Results　(1) Plan: The idea was to establish a frailty prevention class (60 min/session, once a week) consisting of resistance exercises and nutritional or psychosocial programs (standard course, six months, 20 sessions/course; short course, one and a half months, 6 sessions/course; after the course, residents continued with the activities themselves). We planned to launch three classes in the first year (2014) and then to increase the number of classes by ten each year after the second year. (2) Adoption: Out of 154 administrative districts, 36 (23.4%) held frailty prevention classes between 2014 and 2017. (3) Implementation: The median attendance rate for the standard or short course (number of times each participant attended/number of frailty prevention class sessions held) was 75.0%. (4) Reach: A total of 719 older people participated in the standard or short course. The participation rate in the administrative districts, where each frailty prevention class was held, was 32.8%, while at the city level it was 8.1%. (5) Effectiveness: Propensity score matching after multiple imputations were performed. While the prevalence of frailty in non-participants increased by 13.7% in the five years from 2012 to 2017, it only increased by 6.8% in participants. Compared to non-participants, program participants had a significantly lower prevalence odds ratio of frailty at the time of the follow-up survey (OR=0.65, 95% confidence interval 0.46-0.93). (6) Maintenance: After the standard or short course, 25 out of 26 communities (96.2%) continued the frailty prevention activities once a week.Conclusion　The frailty prevention classes were adopted across many districts and lowered the participants' risk of frailty. Moreover, participants continued to engage in frailty prevention activities even after the course. These results indicate the Yabu model's effectiveness and its applicability for a different community.

Clinicopathological characteristics of circumscribed high-grade astrocytomas with an unusual combination of BRAF V600E, ATRX, and CDKN2A/B alternations.

We report four cases of high-grade astrocytoma with a BRAF V600E mutation, ATRX inactivation, and CDKN2A/B homozygous deletion. Children to young adults aged 3-46 presented with a well demarcated contrast-enhancing mass in the supratentorial area. Pathological examination revealed packed growth of short spindle to round polygonal cells including some pleomorphic cells. The tumors had less ability to infiltrate into the adjacent brain parenchyma and presented a circumscribed growth pattern. Mitosis was readily found, accompanied by focal necrosis and/or microvascular proliferation. Tumors were histologically similar in part to pleomorphic xanthoastrocytoma (PXA) or anaplastic PXA, but did not fit criteria for either neoplasm. A BRAF V600E mutation and homozygous deletion of CDKN2A/B were observed, which is similar to the genetic features of PXA or epithelioid glioblastoma, but the additional loss of ATRX nuclear immunoreactivity and absence of TERT promoter mutation were unusual findings, indicating a novel genetic profile. Despite their malignant histological features, all patients had a favorable clinical course and remained alive for 6 months to 28 years under standard medical treatment for malignant glioma. In summary, high grade astrocytomas with BRAF V600E, ATRX, and CDKN2A/B alternations had unique clinicopathological features and may be a novel subset of high grade glioma.

Ectopic thymoma in the paratracheal region of the middle mediastinum: a rare case report and literature review.

BACKGROUND: Thymomas generally arise from the thymus in the anterior mediastinum. Ectopic thymomas arising in the middle mediastinum are rare. We present a case of a thymoma arising from the ectopic thymic tissue in the right paratracheal region. CASE PRESENTATION: The patient was a 67-year-old male who underwent an enhanced-computed tomography examination as preoperative staging for colon cancer. A 20-mm nodule in the right paratracheal region was found incidentally. Fluorodeoxyglucose (FDG) accumulation was detected in this solitary nodule by FDG-positron emission tomography, mimicking an enlarged, possibly malignant lymph node. The tumor was removed by thoracoscopic surgery, and a postoperative pathological diagnosis of type AB thymoma was made. Foci of ectopic thymic tissues were found adjacent to the thymoma. The patient was disease-free and without recurrence 2 years postoperatively. CONCLUSIONS: Including the present case, 13 cases of ectopic paratracheal thymoma have been reported in the English literature, all of which were found on the right side of the paratracheal region. Although ectopic thymomas in the paratracheal region are rare, thymomas may be considered as a differential diagnosis for a paratracheal nodule.

Gliosarcoma with primitive neuronal, chondroid, osteoid and ependymal elements.

A 51-year-old man presented with a 2-week history of malaise. MRI revealed a large solid and cystic lesion with ring enhancement measuring 6.5 cm in diameter in the right frontal lobe. Histologically, the tumor consisted of various components: diffuse growth of atypical astrocytic cells consistent with glioblastoma, fascicular proliferation of atypical spindle cells such as fibrosarcoma, clusters of primitive neuronal cells, and foci of ependymal cells. The sarcomatous component also focally exhibited chondroid and osteoid differentiation. Immunohistochemically, tumor cells in the primitive neuronal component were immunoreactive for synaptophysin and CD56. The spindle cells were immunopositive for Slug and Twist, regulators of epithelial-mesenchymal transition. Direct DNA sequencing demonstrated C228T mutation in the TERT promoter in astrocytic, sarcomatous and primitive neuronal components, suggesting their identical origin. Although a few cases of gliosarcoma with primitive neuronal differentiation have previously been described, the finding that neuronal, glial and sarcomatous components share an identical mutation of the TERT promoter has not been reported. The tumor recurred at the original site 11 months after the first surgery. Interestingly, the recurrent tumor was composed exclusively of a glioblastomatous component, unlike past cases of recurrent gliosarcoma.

CNS high-grade neuroepithelial tumor with BCOR internal tandem duplication: a comparison with its counterparts in the kidney and soft tissue.

Central nervous system high-grade neuroepithelial tumors with BCOR alteration (CNS HGNET-BCOR) are a recently reported rare entity, identified as a small fraction of tumors previously institutionally diagnosed as so-called CNS primitive neuroectodermal tumors. Their genetic characteristic is a somatic internal tandem duplication in the 3' end of BCOR (BCOR ITD), which has also been found in clear cell sarcomas of the kidney (CCSK) and soft tissue undifferentiated round cell sarcomas/primitive myxoid mesenchymal tumors of infancy (URCS/PMMTI), and these BCOR ITD-positive tumors have been reported to share similar pathological features. In this study, we performed a clinicopathological and molecular analysis of six cases of CNS HGNET-BCOR, and compared them with their counterparts in the kidney and soft tissue. Although these tumors had histologically similar structural patterns and characteristic monotonous nuclei with fine chromatin, CNS HGNET-BCOR exhibited glial cell morphology, ependymoma-like perivascular pseudorosettes and palisading necrosis, whereas these features were not evident in CCSK or URCS/PMMTI. Immunohistochemically, diffuse staining of Olig2 with a mixture of varying degrees of intensity, and only focal staining of GFAP, S-100 protein and synaptophysin were observed in CNS HGNET-BCOR, whereas these common neuroepithelial markers were negative in CCSK and URCS/PMMTI. Therefore, although CNS HGNET-BCOR, CCSK and URCS/PMMTI may constitute a group of BCOR ITD-positive tumors, only CNS HGNET-BCOR has histological features suggestive of glial differentiation. In conclusion, we think CNS HGNET-BCOR are a certain type of neuroepithelial tumor relatively close to glioma, not CCSK or URCS/PMMTI occurring in the CNS.

Establishing a new appropriate intramuscular injection site in the deltoid muscle.

It is becoming increasingly important for clinicians to identify a safer intramuscular (IM) injection site in the deltoid muscle because of possible complications following the vaccine administration of IM injections. We herein examined 4 original IM sites located on the perpendicular line through the mid-acromion to establish a safer IM injection site. Thirty healthy volunteers participated in this study and the distances from our 4 IM sites to some anatomical landmarks on their left arms were measured. Ultrasonography (US) was also performed to measure the thickness of the deltoid muscle and identify the posterior circumflex humeral artery (PCHA) along the course of the axillary nerve. Subcutaneous thickness was measured using 2 methods: measuring the skin thickness with caliper after pinching the skin, and with US. The results obtained revealed that the intersection between the anteroposterior axillary line (the line between the upper end of the anterior axillary line and the upper end of the posterior axillary line) and the perpendicular line from the mid-acromion was the most appropriate site for IM injections because it was distant from the axillary nerve, PCHA, and subdeltoid/subacromial brusa. At this site, depth of needle insertions was 5 mm greater than the subcutaneous thickness at a 90° angle, which was sufficient to penetrate subcutaneous tissue in both sexes. Subcutaneous thickness can be assessed with almost the same accuracy by US or measuring with calipers after pinching the skin. The results of the present study support the improved vaccine practice for safer IM injections.