ARG1. Importantly, we found that each case included elevated coagulopathy on laboratory testing and discussed one case in particular with manifestation of bleeding. When diagnosed early, treatment is favorable and can prevent progressive decline. While many states have added ARG1‐D to their expanded newborn screening panels, still many states and countries do not screen for ARG1‐D, and it can be missed in a healthy newborn. We aim to bring awareness to not only the classic presentation as a necessary consideration for otherwise unexplained spastic diplegia but also to the varied presentations of ARG1‐D.Arginase deficiency (ARG1‐D) is an autosomal recessive inborn error of metabolism that is often misdiagnosed. Classic presentation of ARG1‐D includes progressive symptoms of spasticity, delayed development, cognitive impairment, protein avoidance, and seizures. Patients who present atypically may evade diagnosis and require a thoughtful diagnostic workup. Here, we discuss three females of Latin American origin with differing clinical presentations, but who all have the same intronic pathogenic variant in  In contrast to other UCDs, ARG1‐D is distinguished by less frequent and less severe episodes of hyperammonemia that present with chronic toxic effects rather than acute decompensation.Arginase deficiency (ARG1‐D) is a rare genetic inborn error of metabolism (IEM). It is estimated to occur in approximately 1 in 350 000–1 000 000 births,If not identified via newborn screening (NBS), the disease typically presents in early childhood around 3 years of age.We aim to add to the literature on ARG1‐D by demonstrating the contrasting presentations of three diverse patients with ARG1‐D: one with a classic presentation and two with atypical presentations. These cases expand known clinical variability and raise larger concerns about detection and testing strategies in various populations.22.1The study was exempt from IRB approval based on the format of the research. Our patients gave informed consent to have information reported in this study. Existing patients in the literature were reported to have informed consent at the time of the study.2.1.1A 5‐year‐9‐month‐old El Salvadorian female presented to Medical Genetics after referral from Orthopedics for evaluation of weakness, balance issues, and gait abnormalities including increased falling and dragging her feet while walking starting at age 3. She was born in El Salvador to a 29‐year‐old G2 P1 mother and 29‐year‐old father. Pregnancy was complicated by a maternal chikungunya infection at 6 months gestation and she was born full‐term without complication. She was healthy with most developmental milestones achieved at typical times. She had speech delay with vocabulary of 6–10 words at age 3 years. At that time, they noted cognitive decline with difficulty speaking and loss of previously known words. She also had limited activities of daily living including trouble with toileting and dressing herself due to imbalance. She avoided meat and eggs in her typical diet. Family history was unremarkable, with a healthy full sibling.Physical examination demonstrated difficulty with cooperation and verbal expression as well as a small, frail appearance for her age. Her weight was in the 25–50th percentile while her length and head circumference were at the 3rd percentile. She had protuberant, low‐set ears. She was hypertonic in the gastrocnemius and adductor muscles. She had a broad circumduction gait with bilateral foot drop and minimal knee flexion while walking, sustained clonus at the ankle, and hyperreflexia at the patella bilaterally.ARG1 c.466‐1G>C, which is reported by HGMD Professional 2020.3 to disrupt the highly conserved acceptor splice site. She also had a maternally inherited ARG1 mutation with a 32 bp deletion and 21 bp insertion (c.314_345delinsATGCAAGCATCAGGATGACT). While the genetic report classified this mutation as a VUS, its pathogenicity can be deduced due to its causation of disease and inherently large mutation.Due to the concern of an underlying genetic etiology, clinical exome sequencing was performed and revealed a paternally inherited pathogenic splicing variant of She had elevated arginine at 559 μmol/L (41–114 μmol/L) on plasma amino acids (PAA) confirming the ARG1‐D diagnosis. Other essential amino acids were low, indicative of protein‐avoidance. She also had borderline elevated guanidinoacetate (GAA) of 1333 (21–1324 nmol/mL), elevated AST of 133 U/L (29–39 U/L), ALT of 289 U/L (8–24 U/L), and ALP of 404 U/L (117–311 U/L) suggestive of liver dysfunction. She also had an elevated prothrombin time (PT) of 14.5 s (9.0–11.5 s) and an international normalized ratio (INR) of 1.5 (0.9–1.1).When these results were disclosed, the patient was age 6 years old and had continued progression of her symptoms. Her weakness and hypertonia had worsened, particularly in the knees and ankles and she required a walker when ambulating. She also reported new onset fecal incontinence.2/day divided TID. After 2 weeks of strict adherence to the low‐protein diet and beginning medication, she had a pronounced reduction in arginine levels from 566 to 381 μmol/L. At her 3 month follow‐up visit, she had improvement in speech, school performance, and bowel/bladder control. There were no improvements in gross motor activities. She had reported several episodes of seizure‐like activity in a day, that then resolved, and is pending neurology follow‐up.Once ARG1‐D was confirmed, she was guided by a metabolic dietician and started on a low‐protein diet including essential amino acid medical food; she was decreased from 1.3 g/kg/day at baseline to 1 g/kg/day 40% intact protein and 60% essential amino medical food. She was also treated with a nitrogen scavenging medication, glycerol phenylbutyrate 4.5 mL/m2.1.2A 4‐year‐old Honduran female was originally seen by a Medical Genetics inpatient when she was found to have hyperammonemia after presenting with a two‐month history of increased frequency of seizures and a two‐week history of decreased oral intake and vomiting. She was born in Honduras to a G2 P1 25‐year‐old mother and 33‐year‐old father with pregnancy complicated by two bouts of dengue fever. She was born full‐term with typical resuscitation. At 2 months, she had normal development with the gain of head control but then acquired viral meningitis with infarcts to the major cerebral artery demonstrable on magnetic resonance imaging (MRI) performed during the hyperammonemic episode. Subsequently, she lost head control, developed spastic quadriplegia, and seizures and was diagnosed with cerebral palsy. She was also nonverbal and had a history of FTT. Family history was noncontributory. She has two healthy siblings.On physical exam, she measured below the 3rd percentile for both weight and height and in the 50th percentile for a 2.5‐year‐old. Her head circumference was well below the 3rd percentile measuring in the 50th percentile for a 12.5‐month‐old. She appeared generally thin and malnourished, globally delayed, and in mild distress. She was microcephalic with a low anterior hairline. She was wheelchair dependent. She had opisthotonic posturing with extended extremities and contractures, scoliosis, a sacral dimple, and overlapping toes on the left foot. Her neurologic exam revealed hypertonicity throughout, hyperreflexia, and no significant muscle strength against counterforce greater than gravity. She was nonverbal and unable to follow commands.ARG1 (c.466‐1G>C), interrupting a known splice acceptor. MRI of her brain showed extensive encephalomalacia from prior bilateral middle cerebral artery infarcts. Pelvis X‐ray showed a bilateral hip dislocation.Initial laboratory workup was positive for elevated liver enzymes: AST of 204 U/L (0–65 U/L), ALT of 154 U/L (0–37 U/L), and ALP of 524 U/L (142–336 U/L). She also had elevated PT of 16 s (14.1 s) and INR 1.31 (0.85–1.17). She had repeated hyperammonemia of 261, 145, and 126 μmol/L (<45 μmol/L), and urinalysis demonstrating urinary tract infection. PAA demonstrated significantly elevated arginine at 4x the upper limit of normal (477 μmol/L). Cerebral Palsy Spectrum Disorder gene panel showed homozygous pathogenic variants of The hyperammonemia was treated and resolved with the initiation of intravenous fluids containing 10% dextrose. Afterward, she was started on a low‐protein diet with essential amino acid medical food at 2 g/kg/day of protein at baseline decreased to 0.7 g/kg/day, at a ratio of 40% intact protein and 60% essential amino acid medical food. She was also started on nitrogen scavenging medication sodium phenylbutyrate at a rate of 250 mg/kg/day divided TID. A gastrostomy tube was placed to help with the poor intake.After 2 years of treatment, the patient is clinically stable. No functional improvement was evident, but the family reports ease of mobility in and out of the wheelchair with less frequent crying. Plasma arginine decreased post‐treatment but remains elevated to . Her liver enzymes and PT normalized.2.1.3An 8‐year‐old El Salvadorian female presented to the hospital with new‐onset seizures and recurrent nosebleeds. Genetics was consulted for suspicion of vitamin K receptor deficiency after prolonged PT was found that did not improve with vitamin K administration. She was born in the United States to a G2 P1 26‐year‐old mother and 29‐year‐old father at term with no known birth or pregnancy complications. She had a normal NBS performed in the state of Texas. Family history was positive for consanguinity, as the maternal and paternal grandmothers were first cousins. She had three healthy siblings. Her past medical history was significant for developmental delay, FTT, and nosebleeds. She had delayed walking at 2 years 4 months and delayed speech at 2 years. Otherwise, she was reported to be generally healthy with no motor deficits, spasticity, or prior seizures.On physical examination at age 8, her weight and height were less than 3rd percentile , and head circumference was at the 25th percentile. She looked younger than stated age but was generally non‐dysmorphic, and had normal tone, reflexes, and neurologic exam.Imaging of the brain including head CT scan and MRI were unremarkable. An electroencephalogram at initial presentation suggested potential epileptic activity, though no further seizures ensued and no diagnosis of epilepsy was made. She had a normal liver ultrasound and normal skeletal imaging; however, she had delayed bone age.Labs at presentation included elevated transaminases peaking at 219 U/L for AST (0–65 U/L) and 218 U/L for ALT (0–37 U/L), respectively. Labs also showed an elevated PT of 21.5 s (12.0–14.7 s) and INR of 2.25 (0.85–1.17). She also had low Factor VII, IX, and X with an elevated Factor VIII and vitamin K. She did not respond to vitamin K administration. To evaluate for vitamin K receptor deficiency, along with her other medical problems, exome sequencing was recommended but could not be performed due to insurance issues. Her genetic workup was instead performed over time based on what testing would be covered. ARG1‐D was not considered due to lack of spasticity. In turn, she was not diagnosed with ARG1‐D for 2 years, once Whole Exome Sequencing (WES) was covered by her insurance and completed.ARG1 is found on chromosome 6 (OMIM). WES was obtained nearly 2 years after initial presentation and showed a homozygous inheritance of pathogenic variant c.466‐1G>C at ARG1. Her definitive diagnosis became ARG1‐D and standard of care was implemented. Baseline PAA profile demonstrated several low essential amino acids on and plasma arginine level 5x the upper limit of normal at 607 μmol/L.Throughout 2 years of follow‐up with Medical Genetics while attempting to authorize WES, other labs included karyotype, chromosomal microarray (CMA), and gene panels for seizures and glycogen storage disorders which were non‐elucidative. The patient's CMA revealed long contiguous regions of homozygosity in chromosomes 5 and 6 consistent with previously reported consanguinity. Of note, 2/day divided TID. Unfortunately, despite treatment adjustments, her arginine has been difficult to control due to poor acceptance of medical food and continued protein avoidance, resulting in catabolism. During the first several months on a modified diet and prescription nitrogen scavenger treatment, her arginine decreased to mid‐300 s, but her essential amino acids also remained low despite modifications to treatment. Her most recent arginine levels after approximately a year and a half of treatment remain in the upper 400 s to lower 500 s.Diet modification included a low‐protein diet with essential amino acid medical food. Her baseline was 0.95 g/kg/day and she is prescribed 0.95 g/kg/day at a ratio of 50% intact protein and 50% essential amino acid medical food, however, her actual intake is approximately 0.92 g/kg at a ratio of 75% intact protein and 25% medical food. Nitrogen scavenger medication glycerol phenylbutyrate was also initiated at 6 mL/mWhile arginine remains elevated, the patient's clinical presentation has improved significantly over 1.5 years of treatment. Food avoidance and vomiting lessened, improving her quality of life, and her oral intake and growth increased. Her height has improved +0.5 z‐scores. No further hospitalizations or seizures were noted. Cognitive impairment remained, though post‐treatment the impairment did not worsen. She has continued to have no physical impairments.3The three highlighted patients represent contrasting phenotypic presentations in an underrepresented patient population, ranging from classical to coagulation abnormalities without spasticity. Our first two patients demonstrate a common pitfall of ARG1‐D phenotypically appearing as CP. These cases highlight the importance of a thorough workup of spastic diplegia phenotypes, specifically the implementation of ARG1‐D screening, and consideration of metabolic testing in atypical presentations.Diagnosis can be difficult in ARG1‐D, especially in the setting of overlapping and atypical phenotypes. PAA labs are an important screening tool for patients presenting with otherwise unexplained CP. All three of our patients presented with arginine levels 4x or greater than the upper limit of normal, though any increase 3x normal should raise suspicion for ARG1‐D.When clinical and biochemical features suggest ARG1‐D, genetic testing can be used as confirmation. ARG1‐D displays autosomal recessive inheritance, thus biallelic variants are required for diagnosis. As is expected in enzymatic deficiencies, affected ARG1‐D individuals are found to have a genotype–phenotype correlation such that a decreased amount of residual enzyme results in a more severe phenotype and neonatal onset. Many nonsense mutations are thus considered severe,ARG1 variant confirming diagnosis of ARG1‐D. While often ARG1‐D can be misinterpreted as CP, it is therefore possible that the two exist concurrently. It is also likely that the preceding CP diagnosis delayed the discovery of ARG1‐D. Importantly, her vomiting, food avoidance , and hyperammonemia helped suggest a UCD; while spasticity narrowed to ARG1‐D specifically. PAA lab testing was key in leading to the diagnosis of this patient.The first case presented with ARG1‐D in a classic manner, but the second and third cases highlight the clinical variability of manifestations and hurdles in diagnosis. The second patient case included a CP‐like picture that appears to be from viral meningitis in conjunction with ARG1‐D. Her initial diagnosis of ARG1‐D was not evident and she had a typical birth without a traumatic birth event. A case of meningitis subsequently caused multiple brain infarcts evident on MRI. Without records from Honduras, it is impossible to know the causation of her clinical picture; and if hyperammonemia secondary to viral illness played a role in brain damage. The patient did have minor clinical improvements on diet, and plasma arginine and PT lowered. Interestingly, this patient suffered from both a known diagnosis of CP caused by brain assault from meningitis, but also a later‐discovered The third patient presented many unusual phenotypes with both seizures and bleeding dysfunction. Interestingly, she did not have spasticity; but did have a history of intellectual disability and slow growth velocity, which are all associated with ARG1‐D. Although seizures are said to occur in approximately half of patients with ARG1‐D, often without hyperammonemia,All three patients benefited from low‐protein diets demonstrated as decreased arginine and clinical improvement. All had improved lab values and clinical improvement of symptoms at varying degrees, namely with the prevention of hyperammonemia. Additionally, oral nitrogen scavenging medications provided an alternative disposal pathway for nitrogen in amino acid breakdown. Arginine is rich in guanidino nitrogen bases, making nitrogen‐scavenging medications helpful. While the standard of care has shown improved prognoses for patients with ARG1‐D, there remains room for improvement in treatment. Clinical trials for other treatment options, such as enzyme replacement therapy, are underway for this reason. These patients highlight that treatment does have a clinical impact on patients and early diagnosis is the key to initiating treatment prior to hyperammonemia and irreversible damage.Moreover, all three cases emphasize the importance of NBS. Two of our patients were born outside of the US, and another was born prior to ARG1‐D being included on the Texas NBS. As such, none of our patients received NBS that included analytes for ARG1‐D, and thus, diagnoses were missed until well into childhood. As of 2022, 33 United States/territories have included ARG1‐D on NBS,Interestingly, all our patients presented with Central American ancestry. Two were born in other countries, El Salvador and Honduras, and the third was born in Texas to parents who emigrated from El Salvador. They all share the intronic pathogenic variant, c.466‐1G>C. Diez‐Fernandez additionally reports this variant in their cohort study and cites Hispanic and Guatemalan heritage.In summary, prompt diagnosis of ARG1‐D, even in atypical presentations, is paramount in an effort to reduce arginine toxicity with effective treatment methods. Untreated ARG1‐D leads to toxic metabolic byproducts, with subsequent GDD and neuromuscular problems. It is important to remember ARG1‐D as a differential when spastic diplegia, seizures, intellectual disability, FTT, and GDD are present. Otherwise, unexplained coagulopathy may also be caused by ARG1‐D. Prognosis is best when caught early, ideally on an NBS, and additionally necessary for special populations in detecting patients before irreversible damage occurs. Further studies could examine larger cohorts of patients, particularly those with Central American heritage and atypical presentations. Based on the known treatment, positive outcomes, and variability, ARG1‐D is a key diagnosis to garner pediatric clinical improvements and further diet modifications and treatments could improve outcomes for this rare disorder.None.Dr. Farach has worked as a consultant for Aeglea BioTherapeutics in the past. Shelby L. Mills, Paige Roberts, Myla Ashfaq, Kathryn Leal, Hope Northrup, Deborah L. Brown, and David Rodriguez‐Buritica declare that they have no conflicts of interest to disclose.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000 (5).Informed consent was obtained from all patients for being included in the study. Additional informed consent was obtained from all patients for which identifying information is included in this article.


In the article entitled “Indocyanine green fluorescence: A surgeon's tool for the surgical approach of gallstone ileus” that was previously published in Volume 10 Issue 5 of Clinical Case Reports, the author's list was incorrect in the published version.The correct author list should be as follows: Ossian Fuentes, Nicolas Arredondo, Ivan David Lozada‐Martinez, Daniela Guardo‐Carmona, Alexis Rafael Narvaez‐Rojas, Luis Felipe Cabrera‐Vargas.We apologize for the error.


Resectable pancreatic cancer (RPC) is potentially resectable on admission, and the impact of neoadjuvant therapy on these tumors is controversial. Moreover, the safety and efficacy of neoadjuvant chemoradiotherapy with moderately hypofractionated intensity‐modulated radiation therapy (NACIMRT) for RPC have not been studied. Here, we conducted a phase II study to evaluate the safety and efficacy of hypofractionated NACIMRT for RPC.A total of 54 RPC patients were enrolled and treated according to the study protocol. We used moderately hypofractionated (45 Gy in 15 fractions) IMRT with gemcitabine to shorten the duration of radiotherapy and reduce gastrointestinal toxicity. The primary endpoint was overall survival (OS), and we subsequently analyzed the microscopically margin‐negative resection (R0) rate, disease‐free survival (DFS), and histologic effects and safety of NACIMRT.Median OS for the cohort was 40.0 months. Forty‐two patients (77.8%) underwent pancreatectomy after NACIMRT. Median DFS was 20.3 months. The R0 resection rate was 95.2% (40/42) per protocol and 85.2% (46/54) for the cohort. There were no intervention‐related deaths during the study period. Local treatment response, as assessed by the CAP classification, showed no residual tumor in 4.8% of patients. Overall, 23.9% of patients experienced CTCAE grade 3 or 4 during NACIMRT. Adjuvant therapy was initiated in 88% of patients undergoing resection. Postoperative complications grade ≥3b on the Clavien–Dindo scale occurred in 4.8% of patients. CA19‐9 level at enrollment was an independent prognostic factor for OS and DFS.This is the first prospective study of hypofractionated IMRT as neoadjuvant therapy for RPC. Hypofractionated NACIMRT for RPC could be safely introduced with a high induction rate of adjuvant chemotherapy, with an overall survival of 40.0 months. The median overall survival of resectable pancreatic cancer patients after neoadjuvant chemoradiotherpy with intensity‐modulated radiation therapy was 40.0 months without severe toxicity. IMRT has been used for prostate and nasopharyngeal cancers, but precise irradiation is difficult because intra‐abdominal tissues move with respiration. In addition, there is little evidence to support the efficacy of IMRT for pancreatic cancer. Recently, moderately hypofractionated irradiation has been employed for breast22.1This study was conducted as a prospective phase II study of neoadjuvant chemotherapy with IMRT plus gemcitabine (jRCT1051190064) for RPC. The primary endpoint was overall survival (OS), and the secondary endpoints were the microscopically margin‐negative resection (R0) rate, disease‐free survival (DFS), and histologic effects and safety of NACIMRT. All patients diagnosed with RPC according to the NCCN 2016 guidelines18F]fluoro‐D‐glucose (FDG‐PET), and magnetic resonance imaging (MRI) with contrast medium of gadolinium‐ethoxybenzyl diethylenetriamine pentaacetic acid (EOB‐MRI); no pretreatment for current pancreatic cancer; and no hematologic or major organ dysfunction.Inclusion criteria for this study were RPC patients with no arterial tumor contact with the CA, SMA, or CHA, and no tumor contact with the SMV or portal vein or contact of not more than 180° without venous contour irregularity on contrast‐enhanced MDCT. Other inclusion criteria were as follows: histologically confirmed pancreatic ductal adenocarcinoma; age between ≥20 and <80 years; ECOG performance status of 0 or 1; no distant metastases in the thorax, abdomen, or pelvis on dynamic contrast‐enhanced MDCT, positron emission tomography with 2‐deoxy‐2‐[Exclusion criteria were as follows: interstitial pneumonitis; history of irradiation to the upper abdomen; severe comorbidities ; moderate or severe ascites or pleural effusion; history of active cancer ; or expectant or nursing women. The Kyoto University Ethics Committee approved this study (YC1171), and each patient gave informed consent prior to participation.2.22 three times  before chemoradiotherapy. At the start of radiotherapy, gemcitabine was administered at a dose of 1000 mg/m2  concurrently with IMRT. If grade 4 or worse neutropenia or thrombocytopenia occurred, chemotherapy was discontinued for 1 week. For IMRT planning, the gross tumor volume (GTV) included pancreatic tumors and lymph nodes >1 cm in diameter. The clinical target volume (CTV) included the retropancreatic regions between the CA and SMA in addition to the GTV plus a 5‐mm margin, according to our institutional contouring guidelines. The planning target volume (PTV) was defined as the CTV with a 5‐mm margin in all directions. GTV‐PRV and PTV‐PRV were volumes calculated by subtracting the stomach plus 10‐mm and the duodenum plus 5‐mm margins from the GTV and PTV, respectively. Organs at risk of injury were the stomach, duodenum, small intestine, colon, liver, kidneys, and spinal cord, which were delineated on expiratory‐phase CT. The prescribed dose was 45 Gy as D50% of GTV‐PRV and 42 Gy as D95% of PTV‐PRV (Dxx: The dose covering xx% of the target structure) in 15 fractions; PTV D98% ≥36 Gy was preferred.The treatment flow of NACIMRT is shown in Figure Intensity‐modulated radiation therapy was performed with a treatment plan optimized for each patient. The breath‐hold method was used to minimize tumor motion, and cone‐beam CT was used before each treatment to determine daily setup errors. Radiation treatment was delivered using volumetric modulated arc therapy techniques.2.32/day was administered on Days 1–28 of a 42‐day cycle for 6 months as adjuvant chemotherapy, starting 4–8 weeks after resection.Patients were evaluated for feasibility of resection within 4 weeks of neoadjuvant therapy using MDCT, EOB‐MRI, and FDG‐PET. In the absence of unresectable factors such as distant metastases, resection was attempted between 4 and 8 weeks after completion of neoadjuvant radiotherapy. Pancreaticoduodenectomy, distal or total pancreatectomy, and resection of all involved tissues were performed. Operative findings, surgical complications, and histopathology were recorded. S‐1 at a dose of 80 mg/m2.4Resection margins were considered positive (R1) if malignant cells were observed on the surface of the resected specimen (0‐mm margin rule), in the plexus around the SMA or CHA, duodenum, bile duct, or retroperitoneal tissue. If a vein was concomitantly resected, the vein margin was also examined.n = 7). OS was calculated as the time from enrollment to death. Radiological tumor response was evaluated by comparing tumor size on MDCT images before and after NACIMRT according to the Response Evaluation Criteria in Solid Tumors (RECIST v1.0), and pathological responses were evaluated by CAP grade. Toxicity events were recorded using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.Follow‐up data up to December 2022 were analyzed. Patients were evaluated by contrast‐enhanced CT every 3 months for the first 2 years and every 6 months thereafter. A new low‐density mass in the peripancreatic and mesenteric root area was considered a locoregional recurrence. DFS was calculated as the time from the date of resection to recurrence and included off‐protocol patients who underwent resection .The primary endpoint of OS and the secondary endpoints of DFS, response rate, pathologic response, R0 resection rate, surgical morbidity, and acute and late toxicity were evaluated 6 months after the completion of enrollment. The final dataset was carefully reviewed for clerical errors by three physicians . Data for continuous variables were expressed as median and range. Fisher's exact test and the Mann–Whitney 33.1Fifty‐four patients were enrolled in this study. Baseline characteristics of the cohort are summarized in Table 3.2The study diagram is shown in Figure In the group that completed NACIMRT, one patient developed liver metastasis, two patients showed distant lymph node metastasis, and one patient developed peritoneal dissemination; all were treated with chemotherapy. The median CA19‐9 level decreased from 60.45 (0.6–4148.0) U/mL to 23.6 (0.6–2121.0) U/mL, while the NL ratio increased from 2.2 (1.0–4.5) to 3.1 (0.8–24.6) after completion of NACIMRT. The objective radiologic response rate was 28.3%, and median radiographic tumor size decreased from 16.9 (7.0–44.0) mm to 13.0 (7.0–29.0) mm.3.3p = 0.926), blood loss , or postoperative hospital stay  between patients with metal stent and those without. Clavien–Dindo 3a or higher was observed in one case in the metal stent group and in two cases in the non‐stent group, with no significant difference (p = 0.085).The median interval from completion of IMRT to surgery was 26 days . Of the 45 patients who underwent surgery, one had a positive washing cytology, and two had positive distant lymph node metastasis in the para‐aortic region on laparotomy, resulting in 42 patients (77.8%) undergoing pancreatectomy. In the 25 patients with pancreatic head cancer, 12 underwent biliary drainage with metallic stent, with no differences in operative time  started postoperative adjuvant therapy with S‐1 within 6 weeks of resection Table ; 35  of OS was 40.0 months, and R0 was achieved in 95.2% (40/42 patients) of the per‐protocol cohort and 85.2% (46/54 patients) of intention‐to‐treat analysis (ITT) including patients who did not complete NACIMRT. These results are better than those of our previous phase II study of gemcitabine plus IMRT as neoadjuvant therapy for BRPC‐A, in which 93.1% of the per‐protocol cohort achieved R0 resection and 55.3% of patients in ITT.In contrast to postoperative adjuvant therapy with various regimens for RPC, only a few prospective clinical studies have been conducted on neoadjuvant therapy for RPC due to the possibility of tumor resection on admission and concerns about disease progression after neoadjuvant therapy. Nevertheless, several neoadjuvant therapies have been conducted to date to improve survival in RPC patients. In these clinical studies, chemotherapy or chemoradiotherapy has been administrated. In the SWOG S1505 trial, in which FOLFIRINOX or gemcitabine plus nab‐paclitaxel was used as neoadjuvant therapy, 76% of initial patients underwent resection, and 85% of resected specimens were negative for R0 margins. Seventy‐eight percent of patients started and 63% completed adjuvant therapy.This study was conducted with the same purpose as the other studies of improving survival, with some differences in the approach to neoadjuvant therapy for RPC, and our approach has not been previously studied. We used IMRT to deliver therapeutic doses to affected tissue with minimal irradiation to surrounding normal organs compared to conventional radiotherapy. IMRT may have contributed to the low incidence of AEs in this study, resulting in a high completion rate of neoadjuvant chemoradiotherapy . We used moderately hypofractionated schedules, although SBRT (5 fractions) or conventionally fractionated radiotherapy (25–30 fractions) has been used in neoadjuvant radiotherapy for pancreatic cancer.Besides the purpose of reducing toxicity, we used IMRT to increase the irradiation dose to the primary tumor. The biological effective dose at α/β of 10 (BED10) of 45 Gy in 15 fractions was 58.5 Gy, while the BED10 of the conventional standard treatment dose (45–50.4 Gy in 1.8‐Gy fractions) was 53.1–59.5 Gy. Given the shorter treatment duration but higher doses, the treatment intensity of hypofractionated therapy should be higher than that of standard treatment. We calculated the α/β of pancreatic cancer to be 10, which has not been established in the literature. If the α/β of pancreatic cancer were <10, the benefits of hypofractionated therapy would be even greater. Furthermore, the use of IMRT increased treatment intensity compared to the PREOPANC trial, which used conventional 3D conformal radiotherapy with 36 Gy in 15 fractions (BED10: 44.6 Gy). Moreover, our current strategy of increasing treatment intensity while minimizing AEs not only increases the R0 resection rate, but also increases the likelihood of maintaining local control even in inoperable cases, as shown by the favorable local control rate  in our previous study of hypofractionated IMRT for locally advanced pancreatic cancer.Adjuvant therapy with S‐1 is important to improve the long‐term survival of RPC patients. The JASPAC‐01 study with upfront surgery showed better survival  with S‐1 compared to gemcitabine.In this study, elevated CA19‐9 levels (>37 U/mL) at enrollment were independently associated with OS and DFS. Recently, the term bio‐borderline RPC has been proposed to describe tumors with elevated CA19‐9 levels (>37 U/mL) at clinical stages I–II (TNM) that are anatomically resectable but have a poor prognosis.This study has several limitations. First, this was a single‐center study; standardizing IMRT treatment in a multicenter study is difficult because GTV and CTV must be designed as standard procedures according to tumor location, size, and vascular anatomy. Second, the current treatment protocol uses gemcitabine monotherapy as concurrent therapy, which is outdated for the newer doublet or triplet combination therapy. Given the recurrence rate of distant metastases ; data curation ; formal analysis (lead); funding acquisition (lead); methodology ; writing – original draft (lead). Kazuyuki Nagai: Data curation ; formal analysis . Takayuki Anazawa: Data curation ; formal analysis . Yosuke Kasai: Investigation (supporting); validation (supporting). Akitada Yogo: Data curation (supporting); methodology (supporting). Michio Yoshimura: Conceptualization (supporting); methodology ; writing – review and editing (supporting). Takashi Mizowaki: Conceptualization (supporting); methodology (supporting). Norimitsu Uza: Data curation (supporting); validation (supporting); writing – review and editing (supporting). Akihisa Fukuda: Data curation (supporting); validation (supporting); writing – review and editing (supporting). Shigemi Matsumoto: Conceptualization (supporting); writing – review and editing (supporting). Masashi Kanai: Conceptualization (supporting); writing – review and editing (supporting). Hiroyoshi Isoda: Conceptualization (supporting); methodology (supporting). Yoshiya Kawaguchi: Conceptualization ; supervision (supporting). Shinji Uemoto: Conceptualization (supporting); supervision . Etsuro Hatano: Conceptualization (supporting); resources (supporting); supervision .This work was supported by JSPS KAKENHI Grant Number 21K08732.The authors have no competing interests to declare regarding this study.The study protocol has been approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (YC1171) along with all necessary institutional and jurisdictional ethics committees. This study was performed in accordance with the ethical standards of the Declaration of Helsinki and its later amendments. Written informed consent for participation in this study was obtained from each participant.No identifiable data are presented.https://jrct.niph.go.jp/en‐latest‐detail/jRCT1051190064. Registration number: jRCT1051190064. Date of first registration: 06/01/2016.Japan Registry of Clinical Trials (jRCT):


Infective endocarditis should be considered in any febrile individual with acute onset neurological symptoms. If suspicion is high, a negative brain computed tomography does not virtually exclude embolism, and magnetic resonance imaging is warranted.Escherichia coli bacteremia. Computed tomography of the brain was unrevealing; however, scattered embolic phenomena were visualized on magnetic resonance imaging (MRI). The case underscores the importance of clinical awareness, particularly in the setting of unusual microorganisms, and the role of brain MRI in the diagnosis of IE.A diagnosis of infective endocarditis (IE) is often delayed, particularly in those infected with unusual organisms. Hereby, we report a case of a female patient presented with dysarthria, confusion, and altered mental status after being treated for  Brain MRI T2‐FLAIR and DWI showing multiple scattered hyperintense lesions along gray‐white junction bilaterally. She had a past medical history of multivessel coronary artery disease treated with bypass grafting, heart failure with preserved ejection fraction, pacemaker implantation for complete heart block, chronic hypoxemic respiratory failure, diabetes, and chronic kidney disease stage III.3, neutrophil 82.7%) and acute kidney injury . Urinalysis showed significant pyuria with leukocyte clumps, concerning for urinary tract infection. There was no bacterial growth from the urine culture as the sample was obtained 8 h after antibiotic initiation. The first blood cultures were positive for pan‐sensitive Escherichia coli. Initially, she was treated with cefepime 2 grams IV every 12 h for 4 days. Repeat blood cultures became negative, after which were switched to trimethoprim/sulfamethoxazole 800/160 mg orally twice daily. However, she was brought back the following day with confusion, emotional lability, and dysarthria.Laboratory workup showed neutrophilic leukocytosis . Brain MRI T2* did not reveal any cerebral microbleeds. A definite diagnosis of IE was obtained by transesophageal echocardiography (TEE), demonstrating mobile serpiginous mass on the atrial aspect of the P2 and A2 segment of the mitral valve measuring 1.3 × 0.42 and 0.4 × 0.52 cm, respectively . According to the modified Duke criteria,E. coli were not available as the surgery was not performed.Computed tomography of the brain and angiography did not show any evidence of acute stroke or arterial stenosis. Magnetic resonance imaging of the brain was suggestive of scattered embolic phenomena Figure . Brain y Figure . AccordCoxiella burnetii. She was diagnosed with culture‐negative infective endocarditis due to preceding E. coli septicemia and recent antibiotic administration. Cardiothoracic surgery and infectious teams were consulted. Given the high surgical risk and absence of clinical heart failure or severe regurgitation, a prolonged course of intravenous ceftriaxone for 6 weeks was planned.On subsequent hospitalizations, blood cultures drawn at 12 h apart were consistently negative, including antibodies to . Ejection fraction was normal and only trace mitral regurgitation was noted. Again, blood cultures were negative. Ceftriaxone was escalated to cefepime and daptomycin for another 6 weeks. Unfortunately, she was hospitalized again a few days prior to completing antibiotics with sudden onset leg weakness and fall. Repeat brain MRI showed multiple new lesions in juxtacortical left temporal lobe, right cerebellum, and right periventricular white matter. She was evaluated again for the cardiac surgery. In light of overall condition and severely impaired lung function test, a decision was made by the family to pursue hospice care.Two weeks later, she presented with worsening dyspnea, hypoxia, and renal failure, secondary to coronavirus infection. Repeat TTE continued to demonstrate calcified vegetation 2 × 1.5 cm at the tip of the posterior mitral leaflet with mobile component measuring 1.5 × 0.7 cm Figure . Ejecti3Staphylococcus aureus accounted for 26.6% of the cases, followed by Viridans streptococci at 18.7%, group D streptococci at 12.5%, and enterococci at 10.5%.Infective endocarditis is an uncommon life‐threatening infection with an annual incidence of up to 10 per 100,000 of the population.Enterobacteriaceae account for less than 1% of IE cases.E. Coli is a common source of sepsis, it is a much less common cause of IE, accounting for only 0.5%.E. Coli being the most common culprit followed by Pseudomonas aeruginosa.Escherichia coli with phylogenetic type B2, a pathogenic lineage associated with extraintestinal infection, was isolated from an 80‐year‐old woman with emphysematous endocarditis, raising a possibility of certain virulence factors that could have predisposed to endocardial invasion.E. coli endocarditis including end‐stage renal disease, diabetes, malignancies, and chronic alcohol consumption.Despite being previously attributed to intravenous drug use, most non‐HACEK endocarditis cases are more likely to be related to healthcare contact as well as implanted cardiac devices such as prostheses and defibrillators, although the absolute risk is still trivial.Although there is a sevenfold increase in 30‐day mortality if infected leads are retained, removal of implantable devices carries its own risk including cardiac tamponade, stroke, vascular and cardiac injury.Although the incidence of neurological manifestations associated with IE varies among studies ranging from 30% to 47%, obtaining a diagnosis is frequently delayed, resulting in increased morbidity and mortality.Central nervous system lesions involve either a single vessel, particularly the middle cerebral artery, or multiple vessels and may be complicated by purulent meningitis or brain abscess.In a systemic review of patients with IE, brain MRI exhibits greater sensitivity in identifying lesions such as microinfarcts and small hemorrhagic lesions undetected by CT scan.Although the role of brain imaging, either by CT or MRI, is solid in patients with an established diagnosis of IE and neurological symptoms, the role of brain MRI as a diagnostic modality has not been implemented and a decision should be individualized on a case‐by‐case basis. Although urgent surgery is indicated in certain conditions including but not limited to persistent bacteremia despite appropriate antibiotic treatment, hemodynamic instability due to valvular insufficiency, and large vegetation size, the timing for surgery in those with preoperative neurological complications remains ambiguous due to a concern of hemorrhagic transformation.4E. coli has been increasingly reported as a culprit in susceptible patients. In those with neurological symptoms, a negative brain CT does not reliably exclude embolic phenomena and brain MRI should be considered if clinical suspicion remains high.A diagnosis of IE is often delayed or missed, particularly in the setting of bacteremia caused by an unusual organism. Juthipong Benjanuwattra: Conceptualization; writing – original draft; writing – review and editing. Mingxiao V. Yang: Writing – original draft. Amanda L Bell: Writing – original draft. Barbara L Mora: Writing – original draft. Leigh Ann Jenkins: Supervision; validation; writing – review and editing. Pooja Sethi: Conceptualization; investigation; supervision; validation.The authors declare that they have no competing interests.Written informed consent was obtained from the patient to publish this report in accordance with the journal's patient consent policy.


Response rates of immune checkpoint inhibitor (ICI) therapy for recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) are low.This retrospective multicentre cohort study evaluates the predictive and prognostic value of weight loss and changes in body composition prior and during therapy. Patient, tumor, and treatment characteristics of 98 patients were retrieved, including neutrophil and platelet‐lymphocyte‐ratio (NLR and PLR). Programmed death‐ligand 1 (PD‐L1) expression was determined on residual material. Cachexia was defined according to Fearon et al. (2011). Skeletal muscle (SM), visceral adipose tissue (VAT), and subcutaneous adipose tissue (SAT) were evaluated on computed tomography scans at the third lumbar vertebrae level. Univariable and multivariable regression analyses were performed for 6 months progression free survival (PFS6m) and overall survival (OS).p = 0.003), higher baseline SAT index (HR 1.045 [1.02–1.08] p = 0.003), and weight loss <2% (HR 0.85 [0.74–0.98] p = 0.03). Baseline cachexia in combination with >2% early weight loss remained a predictor of OS, independent of PD‐L1 expression .Significant early weight loss (>2%) during the first 6 weeks of therapy was shown in 34 patients (35%). This patient subgroup had a significantly higher NLR and PLR at baseline. NLR and PLR were inversely correlated with SM and VAT index. Independent predictors of PFS6m were lower World Health Organization performance status (HR 0.16 [0.04–0.54] We conclude that the combination of cachexia at baseline and weight loss during ICI therapy is associated with worse OS in R/M HNSCC patients, independent of PD‐L1 expression. The prognostic value of weight loss prior to and during ICI therapy for R/M HNSCC While ICI therapy is standard of care for first‐line therapy of melanoma and non‐small cell lung carcinoma (NSCLC), ICI therapy for recurrent and/or metastatic head and neck squamous cell carcinoma (R/M HNSCC) is still relatively new. Three pivotal phase III trials concerning programmed death ligand 1 (PD‐L1) targeted immunotherapy in R/M HNSCC have been published.Cancer cachexia, a multifactorial syndrome characterized by involuntary weight loss consisting of skeletal muscle and fat mass loss, is a common metabolic problem in HNSCC patients. This is due to the disease itself, to the location of the tumor interfering with adequate caloric intake, and to previous oncological therapy. Cachexia is often accompanied by systemic inflammation, causing a catabolic state that imbalances energy reserves and leads to muscle protein turnover. In turn, this may cause weight loss and muscle mass loss.22.1A retrospective study design was completed according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines.Clinical characteristics including patient, tumor, (previous) oncological treatment characteristics, and the amount of previous palliative systemic treatment lines were retrospectively extracted from the electronic health records. At baseline, the World Health Organization performance status (WHO PS)Long‐term responders were defined as patients receiving ICI therapy for at least 6 months, in other words, patients who had a progression free survival of more than six months (PFS6m) according to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 guidelines.2.22).Abdominal CT scans performed at baseline and at first evaluation as per internal protocol were collected from the database of the radiology department at UZL and MUMC+, and subsequently pseudonymized. Baseline scans were not older than 30 days at start of ICI therapy. The most cranial CT slice on level L3 clearly displaying both vertebral transverse processes was selected for delineation using sliceOmatic software v5.0 (TomoVision). An experienced researcher (over 750 measured CT scans) delineated the areas of interest on the scans and performed the body composition measurements. The observer was blinded to the moment of CT assessment (baseline vs. follow‐up) and to the identity and medical history of the patients. Cross sectional areas (CSA) of SM, VAT, and SAT were measured using pre‐established thresholds of Hounsfield units . SMI, VAT index (VATI), and SAT index (SATI) were calculated using the CSA of SM, VAT, and SAT each divided by height in meters squared  and platelet‐lymphocyte‐ratio (PLR).2.4Representative tumor sections were immunohistochemically stained for PD‐L1 expression using the standardized 22C3 pharmDx assay on the Dako Link 48 platform (Dako). This assay was used as standard in the KEYNOTE‐048.2.5A dedicated head and neck pathologist, certified for PD‐L1 testing, and an experienced head and neck researcher, assessed stained slides. Any discrepancies were resolved through a consensus discussion. Specimens were scored using CPS. This score was defined as the number of positive tumor cells, lymphocytes, and macrophages, divided by the total number of viable tumor cells multiplied by 100. Clinically relevant cut‐offs of ≥1 and ≥20 for CPS were used. Slides that contained less than 100 viable tumor cells were excluded.2.6T‐test and the Mann–Whitney U test, respectively. Categorical variables were analyzed with the Pearson's Chi2 test and Fisher's Exact test where appropriate. Correlations were evaluated using Pearson correlation coefficient. The distributions of OS were estimated by the Kaplan–Meier method and compared by means of the log‐rank test. Cox‐proportional hazard models were used to estimate the hazard ratio (HR) and calculate the corresponding 95% confidence intervals (CI's) for OS. Univariable and multivariable binary logistic regression analysis was performed for long‐term response and autoimmune toxicity. Potential predictive and prognostic variables were selected for multivariable analysis using forward stepping analysis with p for entry ≤0.10 and p to remove upon entry >0.05. Significance was set at the value p < 0.05. Changes in CSA of SM, VAT, and SAT were corrected for days between the baseline and follow‐up CT scan in the regression analyses.Normally distributed variables were reported as means (±standard deviation (SD)). Non‐normally distributed variables were reported as medians (interquartile range (IQR)). Differences between groups were analyzed using independent samples http://vassarstats.net/fisher2×4.html was used.All statistical analyses were performed using SPSS (IBM version 25 for Windows). For the Fisher's Exact test with more than two by two items, the online calculator 3Out of the 177 patients treated with ICI, 98 patients met the inclusion criteria for this study. Information on weight change during the 6 months prior to ICI initiation was available for 87 patients. NLR and PLR could be retrieved for 93 patients. PD‐L1 CPS could be determined in 79 patients.3.1Baseline characteristics are displayed in Table More than half of the population (53%) had low SMI at start of ICI therapy and 39 out of 87 patients with available data on pre‐treatment weight loss were classified as cachectic (45%).3.22), which was also reflected in significantly lower SMI and VATI.During the first 6 weeks of ICI therapy, 34 patients (35%) experienced significant weight loss, defined as more than 2% total body weight loss. When compared to patients with stable or increasing weight during ICI therapy, this subgroup presented a significantly higher NLR and PLR at baseline. Additionally, patients with significant weight loss during the first 6 weeks of ICI therapy had a lower BMI at baseline , but did show a significantly negative correlation with the SMI and VATI . No significant correlation was found between NLR and SATI .Baseline NLR was not correlated with baseline BMI , reflected in correlations with the SMI  and VATI  but again not significantly correlated with SATI .Baseline PLR showed a significant correlation with the baseline BMI (3.3.3Thirty‐three patients (34%) continued treatment for 6 months or longer and were considered as long‐term responders in the current study.p ≤ 0.10) for long‐term response: lower WHO PS, metastatic disease, PD‐L1 CPS ≥1, higher SMI, higher VATI, higher SATI, and absence of significant weight loss during the first 6 weeks of treatment  had deceased. The median follow‐up was 9 months (range 1–64). At 6 months, OS rate was 72.2% and at 1 year 36.7%.p ≤ 0.10) for OS: WHO PS, metastatic disease, PD‐L1 CPS ≥1, second line palliative systemic treatment or higher, weight loss during the 6 months prior to ICI initiation, weight loss categories, catabolic category (explanation below), VATI, and weight loss during the first 6 weeks of ICI therapy.Using univariable Cox regression analysis, the following variables showed a potential predictive value  Cachexia at baseline and stable weight during 6 weeks of ICI therapy, n = 24, (3) No cachexia and weight loss >2% during 6 weeks of ICI therapy, n = 15, and (4) No cachexia and stable weight during 6 weeks of ICI therapy, n = 33. The first category was then defined as the catabolic category including patients with progressive weight loss prior and during ICI therapy. The Kaplan–Meier curve for these cachexia progression categories is shown in Figure As weight loss during the 6 months prior to ICI initiation and during the first 6 weeks of ICI therapy were potential predictors for OS, four categories were created to further elucidate the underlying relationships. The cachexia progression categories are as follows: (1) Cachexia at baseline and weight loss >2% during 6 weeks of ICI therapy, In multivariable forward stepwise Cox regression analysis including all the above‐mentioned potential predictors, WHO PS and the catabolic category remained independent significant predictors for OS in the final model Table . When adp = 0.009), while SAT change and SM change were not significant (data not shown). VAT change did not remain an independent prognostic factor when entered in multivariable forward stepwise Cox regression including the previously mentioned potential predictors from Table To assess which body compartment  contributed most to the prognostic value of early weight loss, regression analysis was repeated for change in body composition corrected for days between the baseline and first follow‐up CT scans. In univariable regression analysis, change in VAT was predictive for OS , thyroiditis (n = 5), colitis or gastritis (n = 3), arthritis (n = 2), pneumonitis (n = 1), and pericarditis (n = 1). Univariable regression analysis to identify potential predictors of autoimmune toxicity revealed a significant predictive value for age with older patients experiencing less immune therapy‐related adverse events (HR 0.92 [0.86–0.99] p = 0.02).Eighteen patients (18%) experienced autoimmune toxicity CTCAE grade 2 or higher within 6 months after ICI initiation. Autoimmune toxicity included dermatitis  during the first 6 weeks of ICI therapy. These patients had a significantly lower BMI at baseline than those with stable or increasing weight. Hypothetically, these patients are in a wasting state that continues during ICI therapy. The low BMI in the subgroup of weight losing patients was reflected in lower values of all three tissue compartments . This subgroup also exhibited a higher NLR and PLR at baseline, as a marker of inflammation. As shown above, patients with baseline cachexia and ongoing significant weight loss during treatment presented with a lower OS. This catabolic category remained a significant prognostic factor, also when corrected for PD‐L1 expression.Strikingly, baseline SMI was not associated with OS, in contrast with a recent publication by Arribas et al.Patients presenting with cachexia at baseline but stable weight during ICI therapy, indicating an arrest in catabolism, showed significantly better survival outcomes. Although baseline cachexia may not predict treatment outcome, the evolution of body weight appears a relevant parameter. Only few studies have reported on ‘early’ changes in body composition during the first weeks of ICI therapy, none in HNSCC patients.Long term responders, with treatment duration of more than 6 months, made up one third of our study population (34%). Reported PFS6m was only 19.7% in the Checkmate 141 study,When focusing on OS, PD‐L1 CPS was not a prognostic factor in this study population as opposed to previous data.4.2Overall, weight loss during ICI therapy remains of prognostic value, more than just baseline cachexia. This weight loss seems to consist of mainly fat mass loss, both VAT and SAT. SAT was found to be an important indicator of clinical outcomes in the current study cohort, which is consistent with the findings of Martini et al.Studies on body composition in cancer patients receiving chemotherapy mainly showed a reduction of muscle mass and function during treatment. The findings concern head and neck cancer, lung cancer as well as other cancer sites.4.3The results need to be considered in the light of a number of limitations. First, accurate body composition evaluation requires CT scans at the level of L3 and therefore patients without baseline and follow‐up CT abdomen were excluded. This could have led to a higher percentage of patients with metastatic disease in the study sample, as these patients received extended CT scans instead of a CT scan of the head and neck region only. Patients with distant metastatic disease receiving ICI therapy had better response in the KEYNOTE‐048 study compared to patients with locoregional recurrence only.Because of the multi‐center study setting, CT scan‐protocols may have differed in slice thickness and dose. Nevertheless, standardized reference points were used for L3 slide selection, and one experienced researcher delineated the structures.Furthermore, the TNM‐classification changed from the seventh to the eighth edition during the study period. In our dataset both the seventh and eighth editions have been used for staging. However, the definition of metastatic HNSCC did not change and tumor stages at the initial diagnosis were not included in the present analysis.Lastly, a trend was observed toward more patients with locoregional recurrent disease experiencing significant weight loss compared to patients with distant metastatic disease. In HNSCC, weight maintenance is particularly challenging due to tumor and previous treatment induced symptoms such as xerostomia, oropharyngeal dysphagia, or odynophagia. The contribution of these factors to weight loss could not be evaluated in this study sample. Retrospective analysis of nutritional interventions was considered unreliable and therefore not included in the analysis.4.4Assessing tumor response is not always clear‐cut based on radiological criteria alone, especially at the first evaluation during ICI therapy. A better understanding of the relationship between a patient's metabolic state and ICI response will help to select patients more accurately and improve the efficacy of ICI treatment in the R/M setting. As such, tracking of weight changes and body composition may prove valuable in the early decision making regarding (dis)continuation of ICI therapy.5The combination of cachexia at baseline and ongoing weight loss during ICI therapy is associated with worse OS in R/M HNSCC patients, independent of PD‐L1 expression, and is predominantly reflected in loss of fat mass. Reversal of weight loss during ICI therapy predicts significant better OS. The underlying mechanisms of continuous weight loss remain unclear. Additional research is needed to define liquid or tumor‐related (inflammatory) biomarkers, identifying the catabolic patient subgroup and additionally pave the way toward improving ICI efficacy.Anna C. H. Willemsen: Data curation ; formal analysis ; investigation ; methodology ; project administration ; validation ; visualization ; writing – original draft . Nina De Moor: Data curation ; formal analysis ; investigation ; methodology ; project administration ; validation ; visualization ; writing – original draft . Jeroen Van Dessel: Investigation ; resources ; writing – review and editing . Laura W. J. Baijens: Supervision ; writing – review and editing . Michel Bila: Writing – review and editing . Esther Hauben: Investigation ; resources ; writing – review and editing . Mari F. C. M. Van den Hout: Investigation ; resources ; writing – review and editing . Vincent Vander Poorten: Resources ; writing – review and editing . Ann Hoeben: Conceptualization ; methodology ; resources ; supervision ; writing – review and editing . Paul M. Clement: Conceptualization ; resources ; supervision ; writing – review and editing . Annemie M. W. J. Schols: Conceptualization ; methodology ; supervision ; writing – review and editing .A.C.H.W., N.D.M., J.V.D., M.B., L.W.J.B., E.H., M.V.D.H., V.V.P., A.H., and A.M.W.J.S. declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. P.M.C. declares the following financial interests which may be considered as potential competing interests: study budget funds from AstraZeneca; was advisory board member for AbbVie, AstraZeneca, Bayer, Bristol Myers Squibb, Daiichi‐Sankyo, Leo Pharma, Merck Serono, MSD, and Vifor Pharma.


To determine obstructive sleep apnea (OSA) phenotypes using cluster analysis including variables of sleep perception and sleep quality and to further explore factors correlated with poor sleep quality in different clusters.This retrospective study included patients with OSA undergoing polysomnography (PSG) between December 2020 and April 2022. Two-step cluster analysis was performed to detect distinct clusters using sleep perception variables including discrepancy in total sleep time (TST), sleep onset latency (SOL), and wakefulness after sleep onset (WASO); objective TST, SOL, and WASO; and sleep quality. One-way analysis of variance or chi-squared tests were used to compare clinical and PSG characteristics between clusters. Binary logistic regression analyses were used to explore factors correlated with poor sleep quality.n = 254), underestimated TST; cluster 2 (n = 158), overestimated TST; cluster 3 (n = 169), overestimated SOL; cluster 4 (n = 155), normal sleep discrepancy and poor sleep quality; and cluster 5 (n = 382), normal sleep discrepancy and good sleep quality. Patients in cluster 2 were older, more commonly had hypertension, and had the lowest apnea–hypopnea index and oxygen desaturation index. Age and sleep efficiency were correlated with poor sleep quality in clusters 1, 2, and 5, and also AHI in cluster 2.A total of 1118 patients were included (81.6% men) with mean age ± SD 43.3 ± 13.1 years, Epworth sleepiness score, 5.7 ± 4.4, and insomnia severity index 3.0 ± 2.4. Five distinct OSA clusters were identified: cluster 1 (Subgroups of patients with OSA have different patterns of sleep perception and quality that may help us to further understand the characteristics of sleep perception in OSA and provide clues for personalized treatment. OSA is associated with an increased risk of cardiovascular, neurocognitive, and metabolic comorbidities . The dias of OSA . A recens of OSA .Sleep discrepancy, defined as the mismatch between subjective and objective sleep data, is an important clinical parameter in both insomniacs and patients with OSA . The difTherefore, although different clustering algorithms have been used in patients with OSA and have included different variables including clinical symptoms, comorbidities, and adherence to positive airway pressure therapy , sleep dThis was a retrospective study of patients with suspected OSA referred to the Sleep Medicine Center, West China Hospital of Sichuan University for polysomnography (PSG) between December 2020 and April 2022. The West China Hospital of Sichuan University Biomedical Research Ethics Committee approved the study (2022–898). Due to the retrospective design, the requirement for informed consent was waived. All data were anonymized and de-identified before release to the investigators.Participants over 18 years of age with the main symptoms of snoring, witnessed apnea, or daytime sleepiness and with polysomnography-derived AHI ≥ 5 events/h were included. Participants with an insomnia severity index (ISI) ≥ 8 and with other sleep disorders such as restless legs syndrome and REM sleep behavior disorder, neurological, or psychiatric disorders were excluded. Patients with other chronic comorbidities that may affect sleep perception and sleep quality, with a TST < 2 h measured by PSG, and with incomplete data were also excluded.Patients underwent full clinical evaluation and had a complete medical history taken. Age, gender, weight, height, body mass index (BMI), smoking and alcohol use, OSA-related symptoms, and comorbidities were recorded. The Chinese version of the Epworth Sleepiness Scale (ESS) was used to assess the subjective daytime sleepiness . The ISIA full-night PSG was conducted for each participant including continuous recordings from an electroencephalogram , electrooculogram (ROC-M1 and LOC-M2), electromyogram , thermistors for nasal and oral airflows, nasal pressure swing, thoracic and abdominal excursion, finger pulse oximetry, electrocardiogram, and body position. Sleep and respiratory events were scored according to American Academy of Sleep Medicine guidelines (v2.3). Apnea was defined as a ≥ 90% decrease in airflow for ≥ 10 s, while hypopnea was defined as a ≥ 30% decrease in airflow for ≥ 10 s associated with an oxygen desaturation of ≥ 3% or an arousal. Total AHI was computed as the sum of apnea and hypopnea events divided by TST. The oxygen desaturation index (ODI) was the number of desaturations ≥ 3% divided by TST.In the morning following overnight PSG, all participants were asked to complete a morning questionnaire that included four questions to assess subjective TST, SOL, WASO, and sleep quality. Subjective TST, SOL, and WASO were measured by open-ended questions and participants were asked to fill in a number. Sleep quality was measured on a Likert scale of “very good,” “good,” “poor,” and “very poor.”As previously, we used differences in subjective and objective TST, SOL, and WASO to reflect sleep discrepancy. A cutoff of 60 min was used for TST discrepancy (TSTdis), with TST underestimation defined as a TSTdis < − 60 min, normal perception as − 60 min ≤ TSTdis ≤ 60 min, and overestimation as TSTdis > 60 min . A cutofP < 0.05.TSTdis, SOLdis, WASOdis, objective TST (oTST), objective SOL (oSOL), objective WASO (oWASO), and sleep quality were used as variables in cluster analysis. As previously, TSTdis and SOLdis are presented as ordinal categorical variables and oSOL, oTST, oWASO, and WASOdis are presented as continuous variables. As the number of patients who rated sleep quality as “very good” and “very poor” were both small, these categories were combined with the “good” and “poor” categories and sleep quality was presented as a binary variable. There were no significant correlations or multicollinearity relationships between variables by Pearson correlation analysis and multicollinearity diagnosis. Moreover, the Kaiser–Meyer–Olkin (KMO) test for factor analysis suitability was < 0.6, indicating that the continuous variables were not suitable for principal component analysis. Therefore, we chose two-step cluster analysis to identify subgroups. After identifying clusters, continuous variables are presented as mean ± standard deviation (SD) and categorical variables as number (percentage). Differences between clusters were analyzed using one-way analysis of variance (ANOVA) or chi-squared tests. Post hoc Bonferroni correction analyses were conducted. Binary logistic regression analyses were performed to explore correlation factors for poor sleep quality in each cluster. SPSS v26.0 was used for all analyses , and statistical significance was defined as 2, while the mean ESS and ISI scores were 5.7 ± 4.4 and 3.0 ± 2.4, respectively. Five distinct and highly discriminant clusters were identified , overestimated TST (cluster 2), overestimated SOL (cluster 3), normal sleep discrepancy and poor sleep quality (cluster 4), and normal sleep discrepancy and good sleep quality (cluster 5). Cluster 2 contained older patients with a higher percentage of OSA-related symptoms and a history of hypertension. Although cluster 2 had the lowest objective sleep efficiency, patients in cluster 1 had more severe AHI, ODI, and mean oxygen saturation. Moreover, the factors correlated with poor sleep quality were different between clusters, with AHI positively and age and sleep efficiency negatively correlated with the risk of having poor sleep quality.TST discrepancy has commonly been used to describe sleep perception . In our Both subjective SOL and SOLdis in cluster 3 were significantly longer than those in other clusters. This would be clinically apparent as subjective difficulty in falling asleep, although the majority of patients in this cluster (60.4%) rated sleep quality as good. Modest overestimation of SOL is common in both insomniacs and healthy sleepers , and thiThe objective WASO was longest and the WASOdis was shortest in cluster 2. Patients in cluster 2 may not correctly perceive wakefulness during sleep and misperceive it as sleep, leading to the perception of normal TST. Although no definite threshold for such wakefulness duration has been established, it was surprising to us that such a long WASO was underestimated, because short wakefulness seems to be more likely to be mistaken for sleep . In addiOur findings also suggested that the subjective sleep quality of patients with OSA could be different even when sleep perception was similar. Moreover, age was negatively associated with a risk of poor sleep in patients with OSA underestimating or overestimating TST. This is different to previous studies reporting that older age is associated with worse objective sleep quality . There mThere are two strengths of our study. First, this was the first cluster analysis identifying OSA phenotypes using sleep perception and sleep quality and we use multiple indicators of sleep perception including SOL, TST, and WASO that make a comprehensive assessment of sleep perception in patients with OSA. Second, we excluded patients with comorbidity of insomnia and other sleep disorders, which eliminated the influence of these diseases on sleep perception and sleep quality.This study has several limitations. First, we only recorded a single night of PSG data and night-to-night variability may have an effect on sleep perception. However, previous studies have demonstrated that night-to-night variability in sleep perception was mainly observed in older patients with insomnia , 30. SecIn this cluster analysis, we obtained five clinical phenotypes of OSA with different sleep perception and sleep quality patterns. Our findings confirmed the heterogeneity and complexity of OSA and may contribute to a deeper understanding of sleep misperception in patients with OSA. The results may also provide clues for the development of more individualized treatment for OSA that will need to be confirmed in future studies.


Microneedles, especially hollow microneedles (HMNs), play an important role in drug delivery, but most of the current HMNs are manufactured based on silicon microfabrication , which are slightly conservative due to the lack of low‐cost, batch‐scale and customized preparation approach, especially for the HMNs with flexible substrate. For the first time, we propose the use of a high‐precision 3D printed master mold followed by a dual‐molding process for the preparation of HMNs with different shapes, heights, and inner and outer diameters to satisfy different drug delivery needs. The 3D printed master mold and negative mold can be reused, thereby significantly reducing the cost. HMNs are based on biocompatible materials, such as heat‐curing polymers or light‐curing resins. The thickness and rigidity/flexibility characteristics of the substrate can be customized for different applications. The drug delivery efficiency of the fabricated HMNs was verified by the in situ treatment of psoriasis on the backs of mice, which required only a 0.1‐fold oral dose to achieve similar efficacy, and the associated side effects and drug toxicity were reduced. Thus, this dual‐molding process can reinvigorate HMNs development. We successfully prepared HMNs with different morphologies, heights and inner and outer diameters. The thickness and rigidity/flexibility characteristics of the substrate can be customized for different applications. When the 3D printed master mold and negative mold were prepared, the total time spent was just tens of minutes to hours depending on the material selected, significantly reducing the cost. To verify the usefulness of this method, psoriasis was generated on the backs of mice. The HMNs required only a 0.1‐fold oral dose to achieve similar efficacy, and the associated side effects and drug toxicity were reduced Figure . Therefo22.1The main HMNs structure consists of patches and microchambers Figure , both ofThe negative mold was filled with liquid biocompatible materials until the liquid surface completely covered the microcolumn array and vacuumed for 10 min until no visible bubbles emerged. Residual bubbles were removed by a dropper, and then the liquid was slowly removed along the edge of the groove until the microcolumn array penetrated the liquid surface, the aspirated liquid can be reused and this step takes only tens of seconds to complete, and the remained liquid leave for 5 minutes to achieve natural leveling. Due to the surface tension, the liquid level was slightly higher at the edge of the groove and the microcolumn array, but this had no effect on the practical application. For the light‐curing polymer, transparent resin with a wavelength of 405 nm, low viscosity and high stiffness was selected and cured by UV light irradiation for 10 min, which was immersed in SDSS solution and uncovered following similar operations to form a light‐curing HMNs patch. The back interfaces were prepared in the same way, aligning and gluing them with the same resin, and thin tubes were used as channels to connect the HMNs to the syringe, forming HMNs on a rigid (resin) substrate. For the heat‐curing polymers, nonphotosensitive polyimide (PI) with a low viscosity and flexibility was selected, and the curing conditions were 140°C for 5 h followed by 240°C for 5 h. The operations were similar except for the curing conditions, allowing HMNs to form on a flexible (PI) substrate, which reduces the cost compared to the traditional process. Parameters such as HMNs shape, height, inner and outer diameter, thickness and the rigidity/flexibility characteristics of the substrate can be customized for different applications. This paper shows 2 types of HMNs, the first having a common shape reported in the literature with heights of 0.8, 1, 1.2, and 1.4 mm, bottom diameters and bottom edges of half the height, rounded (radius = 0.3 mm) at the root of each needle tip to enhance mechanical support. Four commonly used HMNs morphology parameters, square + cone, cylindrical + cone, rhombic and cylinder, were prepared. For square + cone and cylindrical + cone, as the height increases, their inner diameters are 0.2 mm, 0.2 mm, 0.2 mm, and 0.25 mm in order. For rhombic, as the height increases, their inner diameters are 0.25 mm, 0.3 mm, 0.35 mm, and 0.4 mm in order. For cylinder, as the height increases, their inner diameters are 0.35 mm, 0.4 mm, 0.45 mm, and 0.5 mm in order. The first two have a cone and the latter two appear sloped at half of the height to balance drug delivery volume and penetration force.2.2Different needle tip shapes may be suitable for different scenarios, for example, square/cylindrical + cone has sharp tip with slightly smaller hole, which is less likely to be blocked after penetration and has less penetration force, and may be more suitable for surface drug delivery. Rhombic and cylinder has a gentle tip with a slightly larger hole and slightly more penetration force, and may be more suitable for implant drug delivery.2.32, the HMNs required only 1 μg/cm2 MTX to achieve a good therapeutic effect, much lower than the approximately 3.5 μg/cm2 dissolving microneedles,Five‐week‐old male BALB/C mice weighing 20 g were used for in vivo experiments and were divided into 10 groups of 6: normal, model, HMNs + prick only, HMNs + saline, HMNs + methotrexate (MTX) 0.1 mg/kg (dose/weight), HMNs + MTX 0.2 mg/kg, HMNs + MTX 0.4 mg/kg, oral + MTX 0.5 mg/kg, oral + MTX 1 mg/kg and oral + MTX 2 mg/kg. The modeling approach, oral dose grouping and HMNs dose grouping were referenced from the literature.1,b) was used to measure the epidermal thickness, and it was seen that HMNs + 0.2 mg/kg and oral + 2 mg/kg had similar results to the normal group. Mastocyte counting  was used to indicate skin allergies, and HMNs + 0.2 mg/kg had similar results to the normal group, indicating less allergy. The kidney and liver results  showed that there was an increase in the number of blood vessels in all groups except the normal and HMNs + 0.1, 0.2, and 0.4 mg/kg groups, which also indirectly indicated great damage to the liver and kidney. The spleen index  was purchased from Shenzhen Mofang New Material Technology Company. Silicone elastomer PDMS (SYLGARD™ 184) was purchased from Dow Chemical Company. Ecoflex 00‐30 was purchased from SMOOTH‐ON. Light‐curing biocompatible resin (iF 3162) was purchased from Guangzhou Ifun Technology Company.4.2Z‐direction. The resulting samples were removed and soaked with fluoride solution overnight (10 h), blown dry and heated in an oven at 50°C for 2 h to completely cure and enhance the mechanical properties. PDS 2035 equipment was used for the vapor deposition of Parylene C films. Five grams of raw material corresponded to approximately 1 μm thickness, and we used 10 g (2 μm) to completely isolate and protect the sample.The design files were modeled in 3D using a computer and then exported as STL files with an accuracy of 10 μm. Magics Print for BMF 23.2 was used to slice the files layer by layer at 10 μm in the 4.3After the HMNs patch and the back connection structure were prepared, the two were aligned, and a layer of biocompatible resin was applied at the junction as glue. The two were placed under a UV lamp of 405 nm for 5 min to cure, and the excess was cut off with scissors and polished slowly with sandpaper. A TYD02‐02 syringe pump was purchased from Baoding LEADFLUID Technology Company, and the speed was set to 5 μL/min. An infusion tube was attached to the HMNs system in the same way and used to connect the syringe pump. The total elapsed time for single mice was less than 5 min, with good efficiency.4.4Mice were divided into 10 groups with 6 mice per group and managed according to the animal management method of Beijing University of Chinese Medicine, with bedding and food changed daily and ventilation maintained at all times. The hair removal cream Silk & Fresh was purchased from Veet, France, and used for hair removal on the backs of the mice. Sodium pentobarbital (0.3%) was used as an anesthetic, and approximately 100 μL was injected at a time according to body weight. A 5% imiquimod cream was purchased from 3 M Health Care Limited, UK, and used for modeling psoriasis. Methotrexate was purchased from Shanghai ShangPharma Xinyi Pharmaceutical Company for the treatment of psoriasis and was dosed with saline at different concentrations to delineate different experimental groups. Alcohol (75%) and UV disinfection was used to disinfect the environment and HMNs system. Oral administration was performed by professional staff through a gavage needle. The HMNs group was administered by pressing for 1 min after pricking and 2 min of administration time and then pressing for 1 min. Photographs were taken from above by an iPhone 13  at a height of 15 cm.4.5Vernier calipers were used to perform daily double skin thickness measurements on the dorsal surfaces of the mice, and the average was taken after measuring three different locations. An in vivo fluorescence imaging system comprising PerkinElmer's IVIS Lumina SRMS Series product line was used to observe the subcutaneous distribution of rhodamine B in mice. The mice were subjected to an orbital extraction of 1 mL of blood into anticoagulation tubes after removal of the right eyeball. The blood samples were centrifuged at 4°C and 3000 rpm, and serum (300 μL) was separated at high speed for 15 min before liver and kidney function indexes were measured. After blood collection, the mice were fixed in the supine position on a mouse plate, the thorax was quickly opened, excess connective tissue was separated and removed, the spleen was dissected, and the residual blood was gently aspirated from the surface with filter paper. The mice were weighed and photographed and then stored in a −80°C refrigerator. The redundant connective tissues of the mice were separated and removed, the liver was dissected, the residual blood was gently aspirated from the surface with filter paper, the largest lobe of the liver was fixed with 4% paraformaldehyde (PA) to prepare pathological sections, and the rest of the liver tissues were stored at −80°C in the refrigerator. The right kidney was dissected, and the residual blood was gently aspirated from the surface with filter paper. The kidney was cut crosswise, half of the kidney tissue was fixed with 4% PA, and pathological sections were prepared. The whole layer of skin on the dorsum of mice (2 cm × 2 cm) was cut off and fixed with 4% PA to prepare pathological sections. The mouse spleen organs were removed intact and weighed on an electronic balance, and the spleen organ coefficients were calculated according to the formula: spleen organ coefficient = spleen mass (g)/body mass (kg). Pathological tissue changes: liver, kidney, skin behind the ear, and ear tissues were taken and fixed with 4% PA and then made into paraffin sections after dehydration, wax immersion, embedding, and the morphological changes of the tissues were observed by H&E staining. H&E staining was performed by taking paraffin sections of tissues and sequentially performing dewaxing, debenzene, hydration, hematoxylin staining, water washing, and color separation. After the sections appeared pink, were water washed and dyed, the sections were stained with ethanol and dehydrated from low to high concentration, after which the xylene was transparent, and finally the sections were sealed with neutral gum. Pathological sections were observed by randomly selecting five microscopic fields and photographing them at 4× and 10× magnification by technicians who were unaware of the experimental grouping to determine the damage to the liver and kidney tissues and the degree of skin keratinization and inflammatory cell infiltration. The PASI score is an important index for evaluating the severity of psoriasis in clinical and pharmacological studies and includes four aspects: erythema, scaling, infiltration and total score . Erythema, scaling and infiltration were scored as 0, 1, 2, 3, and 4, respectively, according to the severity of psoriasis development, which corresponded to the symptomatology of none, mild, moderate, severe and extremely severe. This scoring was evaluated by three students with different specialties, and the mean value of each index was obtained from the results. A trend line was drawn, thus monitoring the severity of psoriasis in each group of mice.4.6t test. p values less than 0.05 were considered statistically significant and are denoted as follows: ns, no significance, *p < 0.05, **p < 0.01, and ***p < 0.001. All data are plotted as the means ± SDs. All statistical analyses were performed using Origin 2020b (Origin Lab Corporation).Differences between two independent groups were calculated using Yingjie Ren: Conceptualization ; data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); resources (lead); software (lead); validation (lead); visualization (lead); writing – original draft (lead). Junshi Li: Investigation (supporting). Yiwen Chen: Investigation (supporting). Jing Wang: Investigation (supporting). Yuxuan Chen: Investigation (supporting). Zhongyan Wang: Investigation (supporting). Zhitong Zhang: Investigation (supporting). Yufeng Chen: Investigation (supporting). Xiaoyi Shi: Investigation (supporting). Lu Cao: Investigation (supporting). Jiayan Zhang: Investigation (supporting). Huang Dong: Conceptualization (supporting); investigation (supporting). Cong Yan: Investigation (supporting). Zhihong Li: Conceptualization (lead); funding acquisition (lead); supervision (lead); writing – review and editing (lead).The authors have no conflicts of interest to declare.https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/btm2.10530.The peer review history for this article is available at Data S1. Supporting InformationClick here for additional data file.


Saccharomyces cerevisiae. Moreover, we demonstrate the potential of our method to predict unknown transcriptional profiles that would be produced upon genetic perturbation of the GRN governing a two-state cellular phenotypic switch in Candida albicans. We established an iterative refinement strategy to facilitate candidate selection for experimentation; the experimental results in turn provide validation or improvement for the model. In this way, our GRN inference approach can expedite the development of a sophisticated mathematical model that can accurately describe the structure and dynamics of the in vivo GRN.Genetic regulatory networks (GRNs) regulate the flow of genetic information from the genome to expressed messenger RNAs (mRNAs) and thus are critical to controlling the phenotypic characteristics of cells. Numerous methods exist for profiling mRNA transcript levels and identifying protein-DNA binding interactions at the genome-wide scale. These enable researchers to determine the structure and output of transcriptional regulatory networks, but uncovering the complete structure and regulatory logic of GRNs remains a challenge. The field of GRN inference aims to meet this challenge using computational modeling to derive the structure and logic of GRNs from experimental data and to encode this knowledge in Boolean networks, Bayesian networks, ordinary differential equation (ODE) models, or other modeling frameworks. However, most existing models do not incorporate dynamic transcriptional data since it has historically been less widely available in comparison to “static” transcriptional data. We report the development of an evolutionary algorithm-based ODE modeling approach (named EA) that integrates kinetic transcription data and the theory of attractor matching to infer GRN architecture and regulatory logic. Our method outperformed six leading GRN inference methods, none of which incorporate kinetic transcriptional data, in predicting regulatory connections among TFs when applied to a small-scale engineered synthetic GRN in  The establishment of distinct transcriptional programs, where specific sets of genes are activated or repressed, is fundamental to all forms of life. Sequence-specific DNA-binding proteins, often referred to as regulatory transcription factors, form interconnected gene regulatory networks (GRNs) which underlie the establishment and maintenance of specific transcriptional programs. Since their discovery, many modeling approaches have sought to understand the structure and regulatory behaviors of these GRNs. The field of GRN inference uses experimental measurements of transcript abundance to predict how regulatory transcription factors interact with their downstream target genes to establish specific transcriptional programs. However, most prior approaches have been limited by the exclusive use of “static” or steady-state measurements. We have developed a unique approach which incorporates dynamic transcriptional data into a sophisticated ordinary differential equation model to infer GRN structures that give rise to distinct transcriptional programs. Our model not only outperforms six other leading models, it also is capable of accurately predicting how changes in GRN structure will impact the resulting transcriptional programs. These notable features of our model, in conjunction with experimental validation of our predictions in real-world scenarios, contribute to an advancement in the field of gene regulatory network inference. Gene regulatory networks (GRNs) are comprised of interactions between sequence-specific DNA-binding proteins, or transcription factors (TFs), and their respective regulatory target genes . The chaIn the past twenty years, many modeling approaches have been developed to infer GRN architectures using “omics” data , 13–15. Dynamical models suffer from the “curse of dimensionality”, i.e., the problem of a state space or parameter space growing exponentially with the number of genes considered. One approach to dealing with this challenge when building dynamical models is to focus on dynamics near attractors –34, the in silico data and experimental data generated from a real-life GRN constructed using synthetic biology i*, represents the concentrations of the TFs that regulate the ith gene in the GRN, and Anet embodies the structure of a GRN, encompassing the adjacency matrix (AM) and the protein coordination matrix (LG) that govern its behavior. Other symbols in Eqs In these equations, 6:  ifAnethas an independent self activating edgeand the initial system state in which the self activating gene has been set to its maximal expression ∉ n,mIthen7:  append the modified state described above to In,m8:   end if9:  initial system state ← initial system state + mild random perturbation10:  fordo11:  12:   end for13:  record last_statesi,j14:  end for15: 16: forgeneration from 1 to xdo17: fordo18:  mutate AMjby x Hamming distance19:   mutate LGjby x Hamming distance20:   end for21:  fori in 1: mdo22:  initial system state ← In,i23:   update f0for each Anetand add penalty when f0 ∉ 24:   ifAnethas an independent self activating edgeand the initial system state in which the self activating gene has been set to its maximal expression ∉ n,mIthen25:   append the modified state described above to In,m26:    end if27:   initial system state ← initial system state + mild random perturbation28:   fordo29:   30:    end for31:   record last_statesi,j32:   iflast_statesi,jis a fixed point attractorthen33:   record Attractori,j34:    else35:   36:     end if37:   end for38:  39:  ifthen40:  41:   else42:  43:   end if44:  fordo45:  46:   end for47:  48:  49:  end for50: 51: in silico  datasets for method validation, which can provide gene expression data that is directly predicted based on a hypothetical “source” GRN model i,min and [R]i,max are the minimal and maximal expression levels of the ith gene. Table D. Comparison of inference software features. Table E. The in silico test result for protein coordination matrix. Table F. The in silico test result for f0. Table G. Variance of the outcome networks in the in silico test.(PDF)Click here for additional data file.S1 DataTable A. Ground truth for the 5-gene in silico network. Table B. Ground truth for the 6-gene in silico network. Table C. Ground truth for the 7-gene in silico network. Table D. Ground truth for the 8-gene in silico network. Table E. Ground truth for the 9-gene in silico network. Table F. Model comparison results for the in silico GRNs comprising autoregulation. Table G. Model comparison results for the in silico GRNs comprising no autoregulation. Table H. Overall Summary of Predicting Unkown in silico Attractors. Table I. Knockout attractors prediction results of the 5-gene networks. Table J. Knockout attractors prediction results of the 6-gene networks. Table K. Knockout attractors prediction results of the 7-gene networks. Table L. Knockout attractors prediction results of the 8-gene networks. Table M. Knockout attractors prediction results of the 9-gene networks. Table N. Model comparison results for the synthetic GRN built in S. cerevisiae. Table O. Comparison of attractors between our model and Cantone et al.’s model [S. cerevisiae. Table P. The consensus “directed” C. albicans GRN inferred by our model using all 17 attractors. Table Q. Comparison between C. albicans experimental transcriptional profiles and attractors inferred by the consensus “directed” GRN.’s model  for the (XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.