Many plant leaves have two layers of photosynthetic tissue: the palisade and spongy mesophyll. Whereas palisade mesophyll consists of tightly packed columnar cells, the structure of spongy mesophyll is not well characterized and often treated as a random assemblage of irregularly shaped cells.Using micro‐computed tomography imaging, topological analysis, and a comparative physiological framework, we examined the structure of the spongy mesophyll in 40 species from 30 genera with laminar leaves and reticulate venation.A spectrum of spongy mesophyll diversity encompassed two dominant phenotypes: first, an ordered, honeycomblike tissue structure that emerged from the spatial coordination of multilobed cells, conforming to the physical principles of Euler’s law; and second, a less‐ordered, isotropic network of cells. Phenotypic variation was associated with transitions in cell size, cell packing density, mesophyll surface‐area‐to‐volume ratio, vein density, and maximum photosynthetic rate.These results show that simple principles may govern the organization and scaling of the spongy mesophyll in many plants and demonstrate the presence of structural patterns associated with leaf function. This improved understanding of mesophyll anatomy provides new opportunities for spatially explicit analyses of leaf development, physiology, and biomechanics. Tissue samples were enclosed between two pieces of polyimide tape to prevent desiccation while allowing high X‐ray transmittance during the scan. They were mounted in the sample holder, centered in the microCT X‐ray beam, and scanned using the continuous tomography mode, which captured 1025 (ALS) or 1800 (SLS) projection images at 21 keV, using a 5×, 10×, or 40× objective lens, yielding final pixel resolutions between 1.277 and 0.1625 µm. Each scan was completed in 5 min (SLS) or c. 15 min (ALS).MicroCT data were obtained at the Advanced Light Sources (ALS) at the Lawrence Berkeley National Lab  and at the TOMCAT Tomography beamline, Swiss Light Source . Samples were prepared before each scan (< 30 min) by excising a et al.  indicated low intraspecific variance  and spongy mesophyll cell counts (c. 40–140 per sample) were measured from areas between veins. All measurements were obtained using Fiji. To account for the spatial constraints on local mesophyll topology imposed by veins, we measured minimum vein spacing distance, defined as the shortest characteristic distance between veins and measured as the mean length between the highest order veins and closest neighboring veins . Leaves were placed in the chamber and allowed to acclimate to the following conditions until they reached a steady state: photosynthetic photon flux density, 0, 50 100, 200, 400, 600, 800, 1000 µmol m−2 s−1; CO2, 400 ppm; relative humidity, 37%; vapor pressure deficit, 1.6 kPa; leaf temperature, 25°C. Measurements were performed on three or four leaves per species. Published data sources , the total spongy mesophyll volume Vmes (µm3), and the spongy mesophyll surface area exposed to the IAS SAmes (µm2). Mesophyll porosity (m3 m−3) was calculated as the IAS volume fraction of the total spongy mesophyll volume. Spongy mesophyll surface area per unit tissue volume was calculated as the ratio SAmes/Vmes (µm2 µm−3). Mesophyll surface area per projected leaf area Sm (m2 m−2) was calculated as the ratio SAmes/SAleaf (m2 m−2), where SAleaf (m2) is the width multiplied by the height of the leaf tissue in the transverse plane.Image stacks were cropped to spongy mesophyll domains by excluding veins, palisade mesophyll, and epidermal layers, and the airspace was segmented by visually and subjectively defining a range of pixel intensity values that optimized airspace classification while minimizing false classification. The Ivizo 2019.4; Thermo Scientific, Hillsoboro, OR, USA) was used to model the 3D connectivity and geometry of the IAS networks and to model material properties such as tortuosity and flow directionality. Image stacks cropped to isolated spongy mesophyll volumes were imported into the Avizo xporenetworkmodeling extension. IAS connectivity was measured by running the Volume Fraction module on each of the total airspace and interconnected airspace objects. The Generate Properties function was used to approximate vapor‐phase flow through the network using boundary values of 40 Pa and 25 Pa as input and output pressure  of each spongy mesophyll IAS pore in the paradermal plane by first determining the center of each pore, then computing a Voronoi diagram, and subsequently counting the number of sides of the Voronoi cell associated with each pore. Nearest‐neighbor analysis was implemented using the IBrocher, . For imacts Fig. . For fulstats (v.3.6.23) in R  in a large 2D aggregate according to:E surrounding each face F the number of vertices V per face will also be six. This also results in an edge connectivity Ze of three, resulting in a hexagonal honeycomb that tessellates 2D space with the least material investment . The number of edges per face n¯ was calculated from the edge coordination relationship:t‐test was done to test agreement between n¯ and the nearest‐neighbor measurements. A two‐sided analysis (t(55) = 11.2, P < 0.001) showed there was no significant difference between n¯  and nearest neighbors .The topological constraints of Euler’s law  introduces a corresponding polygon with a higher number of edges into the aggregate, frequently as a neighbor. For full details on the theory and methods of tessellation entropy, Lewis’ rule, and the Aboav–Weaire law, see Methods Topological methods can be used to describe how ordered or regular a structure is. We described dispersion in the IAS pore polygon class of the spongy mesophyll using tessellation entropy and by comparison with theoretical predictions from Lewis’ rule and the Aboav–Weaire law. Lewis’ rule of polygon size dispersion is derived from the space‐filling properties of cucumber epithelial cells Lewis, , 1931 ans Lewis, , where trandomforest package . OOB error estimate was 2.5%; that is, 97.5% of samples were correctly classified. The confusion matrix  and its stored family‐level supertree (v. R20120829) using the R package brranching   with other leaf traits , stomatal guard cell length , and the diameter of the IAS pores . These relationships remained significant after accounting for shared evolutionary history . For example, the spongy mesophyll of the two species shown in Fig. B. nervosa and Q. suber, respectively) yet dissimilar IAS organizations.Although the IAS frequently appeared segmented into discrete domains bounded by cells in 2D paradermal and transverse planes of the leaf Fig. , 3D netwAL, cell packing density, and IAS channel radius . These species had relatively small spongy mesophyll cell dimensions and high cell packing densities, yet they exhibited tissue lattices , in which chains of elongated cells attached to veins at right angles, forming an approximately rectilinear lattice  were approximations of dominant patterns in the spectrum of structural organization and do not preclude intermediate forms. Several species had transitional characteristics, for example five species placed into cluster 2 (predominantly nonhoneycomb) by cluster analysis were instead observed to have the honeycomblike phenotype  and AL. We found that, as arm length increased, SAmes/Vmes of the spongy mesophyll decreased sharply according to a power law . Thus, in addition to cell size and cell packing density  = 26.65, P < 0.001). Higher Amax values occurred among species with nonhoneycomb phenotype  = 46.06, P < 0.001); thus, although the palisade mesophyll is typically modeled with a higher photosynthetic capacity relative to the spongy mesophyll .Table S6 Standardized major axis regressions.Click here for additional data file.Video S1 MicroCT volume rendering of a coffee (Coffea arabica) leaf.Click here for additional data file.Video S2 MicroCT volume rendering of a star anise (Illicium floridanum) leaf.Click here for additional data file.Video S3 MicroCT volume rendering of a cork oak (Quercus suber) leaf.Click here for additional data file.Video S4 MicroCT volume rendering of a cotton (Gossypium hirsutum) leaf.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries  should be directed to the Click here for additional data file.


The aim of this study was to assess the effectiveness and tolerability of atezolizumab plus carboplatin and etoposide combination chemotherapy in elderly patients with extensive‐disease (ED) small‐cell lung cancer (SCLC).n = 36 patients; median age: 74 years [range: 70–89 years]) and the non‐elderly group .This retrospective study evaluated 65 SCLC patients who received atezolizumab, carboplatin, and etoposide for ED‐SCLC in nine study institutions between August 2019 and September 2020. Clinical efficacy, assessed according to response rate and survival, and toxicity were compared between the elderly (p = 0.18) and the median overall survival  between the elderly group and the non‐elderly group. The frequencies of grade ≥3 hematological adverse events in the elderly patients were as follows: decreased white blood cells, 36.1%; decreased neutrophil count, 61.1%; decreased platelet count, 8.3%; and febrile neutropenia, 8.3%. One treatment‐related death due to lung infection occurred in the elderly group.The response rate was 73.8% (80.5% in the elderly group and 65.5% in the non‐elderly group). There was no significant difference in both the median progression‐free survival  small‐cell lung cancer (SCLC). There was no significant difference in both the median progression‐free survival  and the median overall survival  between the elderly group and the non‐elderly group. Atezolizumab plus carboplatin and etoposide could be the preferred standard treatment modality for elderly patients with ED‐SCLC. Accordingly, the incidence is also currently increasing in parallel with the gain in life expectancy worldwide. A disproportionate impact on the elderly population has been observed, along with a significant gain in the occurrence of lung malignancy in the older adult. More than 50% of lung cancer patients are diagnosed at an age of 65 or older, the lower limit of what is defined as “elderly” in epidemiological studies.To date, four cycles of carboplatin plus etoposide remain the standard first‐line therapeutic regimen for older adults patients with ED‐SCLC.Atezolizumab is a humanized monoclonal anti‐programmed death ligand 1 (PD‐L1) antibody, an immune checkpoint inhibitor that blocks PD‐L1‐programmed death 1 (PD‐1) and PD‐L1‐B7‐1 signaling and recovers tumor‐specific T‐cell immunity.Although systemic treatment consisting of ICIs plus platinum and etoposide combination therapy is considered to be an effective treatment for ED‐SCLC based on the results of the aforementioned studies, the effectiveness and feasibility of atezolizumab, carboplatin, and etoposide in older adult patients with ED‐SCLC have not been fully assessed. Thus, this current study aimed to evaluate the activity and feasibility of atezolizumab plus carboplatin and etoposide among elderly patients with ED‐SCLC.22.1This study analyzed the records of patients diagnosed with ED‐SCLC who received the therapeutic regimen of atezolizumab plus carboplatin and etoposide between August 2019 and September 2020 in nine Japanese institutions. The design of this study is retrospective. The inclusion criteria were (1) cytological or histological diagnosis of SCLC with stage III/IV with no curative irradiation or postoperative recurrence and (2) first‐line chemotherapeutic regimen with atezolizumab, carboplatin, and etoposide combination chemotherapy. The elderly was defined as patients aged ≥70 years; patients aged <70 years were categorized as non‐elderly. Pathological stage III/IV SCLC was determined based on the Union for International Cancer Control tumor–node–metastasis (TNM) Classification, Seventh Edition.All patients underwent pretreatment physical exam, chest radiography, thoracoabdominal computed tomography, brain computed tomography or magnetic resonance imaging, and2.22 body surface area, intravenous injection on days 1 through 3 of each cycle), followed by atezolizumab maintenance every 3 weeks. Granulocyte colony‐stimulating factor was administered as prophylaxis against neutropenia at the discretion of the attending physician. Treatment was terminated when disease progression was observed, intolerable toxicity occurred, or the patient withdrew consent for treatment.All the patients had no history of atezolizumab, carboplatin, and etoposide combination therapy, and each patient was administered up to four cycles of atezolizumab , carboplatin , and etoposide  or death from any reason. OS was calculated from the initiation of treatment until death or was censored on the day of the last visit. PFS and OS were analyzed with the Kaplan–Meier method and compared between the two groups using the log‐rank test. Differences were considered statistically significant at a two‐tailed p‐value of <0.05. All statistical analyses were performed using the JMP statistical software, version 11.0, for Windows .Categorical variables were calculated using Fisher's exact test, and continuous variables were calculated using Welch's 33.1A total of 98 patients were administered combination chemotherapy with atezolizumab plus carboplatin and etoposide. Among them, chemotherapy was performed as second‐ or subsequent‐line treatment in 17 patients, and atezolizumab was added during the chemotherapy in 16 patients. To maintain uniformity in patient background, 33 patients were excluded. Finally, 65 patients were evaluated; of them, 36 and 29 patients were aged ≥70 and < 70 years, respectively. The patient selection diagram is shown in Figure 2 for etoposide . The other doses were used in only two patients. In the elderly group, the most common doses were also AUC 5 for carboplatin and 100 mg/m2 for etoposide . Dose reductions for etoposide and for both carboplatin and etoposide were made in 6 and 7 patients, respectively.Table 3.2In the overall cohort, the overall response rate (ORR) was 73.8% (95% CI: 61.9–83.0), and the disease control rate was 89.2% (95% CI: 79.1–94.9). Treatment response according to the patient group is shown in Table 3.3p = 0.18). The OS based on age (≥70 years/<70 years) is shown in Figure p = 0.24). In addition, no significant difference was observed in any of the variables in the multivariate analysis of the entire patient population, except PFS according to prior radiotherapy . At that median follow‐up period, the median PFS and OS were 5.4 months (95% CI: 4.6–5.9 months) and 15.9 months (95% CI: 11.8–18.3 months), respectively. In total, 57 (87.6%) patients experienced disease progression and 41 (63.0%) patients died from events related to the primary disease. PFS was similar in both groups Figure . The medp = 0.09, Figure p = 0.90, Figure In the elderly patients, the median PFS was 5.9 (95% CI: 5.2–8.2) months in the patients aged 70–74 years and 4.8 (95% CI: 3.2–6.0) months in those aged ≥75 years  patients with untreated ED‐SCLC, the ORR in the carboplatin plus etoposide group was reported to be 60.0%, with a median PFS of 4.4 months and median OS of 11.3 months.These discrepancies could be attributed to differences in patient backgrounds or other biases. However, our results are comparable to the median OS of 14.6 months in the Japanese subgroup analysis of the IMpower133 study,For further analysis, the elderly patients were divided into two cohorts by age: patients aged 70–74 years and those aged ≥75 years. As a result, no significant differences in the response rate, median PFS, and median OS were observed between the two groups Table . HoweverUnivariate analysis of the data of the patients aged ≥70 years demonstrated that the PFS and OS did not significantly differ according to any of the clinical factors. These results suggest that combination chemotherapy of atezolizumab plus carboplatin and etoposide is universally effective in patients older than 70 years. However, further investigation could not be analyzed due to the small number of patients with a poor PS (≥2). Future studies on the efficacy of atezolizumab, carboplatin, and etoposide in patients with poor PS are warranted.With respect to toxicity events, they were similar between the two cohorts in this study. However, the percentage of patients with decreased neutrophil count was higher in the current study than in the IMpower133 study,However, one patient in the elderly group had treatment‐related death due to bacterial infection. The patient received four cycles of atezolizumab, carboplatin, and etoposide combination chemotherapy and died of pneumonia and lung abscess after three cycles of atezolizumab maintenance therapy. It is likely that incidental infection developed when the patient achieved PR. In addition, the occurrence of toxicities was similar between the elderly and non‐elderly groups, although this was a comparison of a small population. These findings indicate that the adverse event signals of atezolizumab plus carboplatin and etoposide combination chemotherapy may be well tolerated by elderly SCLC patients.Biomarkers such as tumor mutation burden and PD‐L1 protein expression lack accuracy for the identification of SCLC patients who will optimally benefit from atezolizumab plus carboplatin and etoposide combination chemotherapy.There are some limitations in the current analysis. First, this was a retrospective study. In addition, it was an analysis with a small sample size; larger prospective trials are needed to verify the clinical usefulness of our findings. Second, the treating physician's decision was made to reduce, skip, or delay a treatment with anticancer agents. To reduce this bias to the greatest extent possible, all consecutive patients treated at the study sites were enrolled in the analysis, and their clinical records were thoroughly reviewed. Third, the administration of atezolizumab, carboplatin, and etoposide combination chemotherapy for first‐line treatment and the chemotherapeutic regimen for subsequent‐line treatments were determined by the attending physician based on the policy of each institution. These decisions could have introduced selection bias, which is an inherent limitation of retrospective studies. The possibility that this may have affected survival could not be ruled out. The treating physician's decision to reduce, skip, or delay a treatment with anticancer agents.In conclusion, our real‐world data provide evidence that atezolizumab plus carboplatin and etoposide combination chemotherapy might be considered to be a feasible treatment strategy with favorable efficacy for treatment‐naïve elderly patients with SCLC. These findings may provide a new orientation in the pharmacological management of elderly patients with SCLC. In the future, it is necessary to conduct prospective studies on the effects of atezolizumab, carboplatin, and etoposide combination chemotherapy in elderly patients.All authors have read and approved the final manuscript. Conceptualization and methodology, A.S. and H.I.; formal analysis and data curation, H.I. and K.K.; Project administration, visualization, and writing—original draft preparation, A.S. and H.I.; Supervision, K.K. and H.K.; Investigation and resources, S.W., T.T., Y.N., H.M., Y.Y., T.K., Y.U., H.T., O.Y., A.M., H.T., and K.M.; writing—review and editing, all authors.The authors have declared that there are no competing interests.All procedures complied with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The study design was approved by the Institutional Ethics Committee of International Medical Center, Saitama Medical University (No. 2021–113).The requirement for written informed consent was waived by the ethics committee of Saitama Medical University because of the retrospective nature of the study.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


CENPF gene are responsible for Strømme syndrome, a condition presenting with intestinal atresia, anterior ocular chamber anomalies, and microcephaly. Through an international collaboration, four individuals  carrying CENPF biallelic variants, including two missense variants in homozygous state and four LoF variants, were identified by exome sequencing. All individuals had variable degree of developmental delay/intellectual disability and microcephaly (ranging from −2.9 SDS to −5.6 SDS) and a recognizable pattern of dysmorphic facial features including inverted‐V shaped interrupted eyebrows, epicanthal fold, depressed nasal bridge, and pointed chin. Although one of the cases had duodenal atresia, all four individuals did not have the combination of internal organ malformations of Strømme syndrome . Immunofluorescence analysis on skin fibroblasts on one of the four cases with the antibody for ARL13B that decorates primary cilia revealed shorter primary cilia that are consistent with a ciliary defect. This case‐series of individuals with biallelic CENPF variants suggests the spectrum of clinical manifestations of the disorder that may be related to CENPF variants is broad and can include phenotypes lacking the cardinal features of Strømme syndrome.Biallelic loss‐of‐function (LoF) variants in  At birth facial dysmorphisms were noted and on day two of life, he had respiratory distress requiring oxygen. He walked independently at 13 months of life and pronounced his first words at 18 months of age. At the age of 4 years 7 months, he could pronounce 20 words but no complete sentences, and he understood and followed simple commands. On clinical evaluation at 4 years and 7 months of age, his weight was 16 kg (19th centile), his stature was 105.5 cm (39th centile), and his occipito‐frontal circumference (OFC) was 48 cm (−3 SDS). He showed down‐slanting palpebral fissures, deep‐set eyes, flat philtrum, bulbous nasal tip, anteverted nares, low‐set ears, and shawl scrotum . He had increased nuchal translucency on prenatal ultrasound, and at birth had lymphedema of the feet. The lymphedema resolved but returned several years later. After birth, he was admitted for feeding difficulties and multiple congenital anomalies, which included coarctation of the aorta, bicuspid aortic valve, duodenal atresia, horseshoe kidney, intestinal malrotation, and microcephaly. Other features included short stature, failure to thrive, bilateral mild conductive hearing loss, ocular anomalies , vesicoureteral reflux, and cryptorchidism. He developed significant gastrointestinal complications, with recurrent episodes of acute pancreatitis associated with pancreatic stones, and multiple episodes of gastrointestinal bleeding due to duodenal and gastric varices. He also had chronic daily headaches, and a brain MRI at 11 years showed mild decrease in size of the frontal lobes with possible malformation of cortical development. Early developmental history was unremarkable, with no gross or fine motor delays. He had learning difficulties and at the last visit, he was in the fifth grade but functioning on a first or second grade level academically, thus requiring special education classes with additional assistance in mathematics. At 7 years of age, he was noted to have average intellectual functioning on the KBIT‐2 and average/below average range in academic achievement on the WIAT‐III. His dysmorphisms included hypopigmented hair, high nasal bridge, micro/retrognathia, small mouth, pterygium colli, single palmar creases, fifth finger clinodactyly, persistent fetal fingertip pads, and hypoplastic nails  maternal , and NM_016343.3:c.5242G>T:p.(Glu1748Ter) paternal , length of 40 cm (−2.9 SDS), and OFC of 27.5 cm (−2.9 SDS). In the first month of life, he had feeding problems due to partial bowel obstruction, which resolved without any intervention. His motor development was within normal range. Global developmental delay was suspected from 1 year of age and became obvious later; he visited a special needs school. A WISCII showed an IQ of 48. His verbal communication was adequate. He had attention‐deficit hyperactivity disorder. He developed type 1 diabetes mellitus at the age of 6 years. On clinical evaluation at 10 years of age, his height was 137.5 cm (−0.14 SDS), his weight 34.2 kg (0.5 SDS), and OFC was 45.5 cm (−5.6 SDS). He had a sloping forehead, short palpebral fissures, and full lips  . By newborn screening, she was diagnosed with hypothyroidism due to sublingual ectopic thyroid and she has been on hormonal replacement therapy with thyroxine since then. She had feeding difficulties in the neonatal period. At 1 month of age, she developed bronchiolitis and severe upper airway obstruction requiring tracheostomy that was maintained until she was 12 months of age. Her development was delayed: she walked independently by 2 years and said her first words age 4 years. She had moderate‐to‐severe learning difficulties and attended special school. Physical examination at the age 17 years, revealed a height of 150 cm (first centile), weight of 51.6 kg (24th centile), and OFC of 51 cm (−3.3 SDS). She was noted to have microcephaly with receding forehead, highly arched palate, and dental crowding . Following informed consent genomic DNA from the patient and her parents underwent ES, enriched using the SureSelect Clinical Research Exome  and sequences in the NextSeq 500 sequencing system . A custom pipeline based on Burrows‐Wheeler Alignment tool (BWA), Genome Analysis Toolkit (GATK), and ANNOVAR .Case 2Genetic testing was performed in the setting of diagnostic testing. Using genomic DNA from the proband and parents, exon regions and flanking splice junctions of the genome were captured using the Agilent Clinical Research Exome kit . These targeted regions were sequences simultaneously by massive parallel (NextGen) sequencing on an Illumina HiSeq 2000 sequencing system with 100 bp paired‐end reads. Bi‐directional sequence was assembled, aligned to reference gene sequences based on human genome build GRCh37/UCSC hg19 and analyzed for sequence variants using a custom‐developed analysis tool . Sanger sequencing was used to confirm potentially pathogenic variants identified.Case 3ES was performed as routine diagnostics in a clinical setting. Genomic DNA was fragmented into 200–500 bp fragments by means of Adaptive Focused Acoustics  shearing according to the manufacturer's protocol. Exomes were captured using the Agilent SureSelectXT Clinical Research Exome v2 capture library kit  accompanied by Illumina paired end Sequencing on the HiSeq4000 , generating 2 × 150 bp paired end reads with at least 80× median coverage. An in‐house sequence analysis pipeline  based on read alignment using BWA‐(MEM) and variant calling using the GATK Haplotype Caller1 and UnifiedGenotyper2 was used to align reads and call variants on the generated BAM files. Variants were subsequently annotated using the Variant Effect Predictor3. Included annotation fields were, among others, variant consequence, sift scores, polyphen scores, CADD scores, and allele frequencies in the 1,000 Genomes populations. An in‐house developed tool, additionally annotated variants using dbSNP132, gnomAD allele frequencies, and the Genome of the Netherlands (GoNL) frequencies. After annotation, variants with an allele frequency of >5% in the GoNL or in the 1000 Genomes project were excluded from further interpretation. LOVDplus  was used for interpretation of variants.Case 4Following informed consent, the patient was enrolled in the Deciphering Developmental Disorder study, which offers trio ES for children with developmental disorders by the systematic application of the latest microarray and sequencing methods as previously described . When cells attained 90% confluence, they were cultured in serum‐free media for another 48 hr to induce ciliogenesis. Cells were fixed with ice‐cold methanol for 5 min, then permeabilized and immunostained with antibody against the ciliary component ADP‐ribosylation factor‐like GTPase 13B  and the centrosome marker γ‐tubulin . Donkey anti‐rabbit AlexaFluor 488 (A21206) and donkey anti‐mouse Alexa Fluor 568  were used as secondary antibodies. DNA was stained with Hoechst . Samples were examined under LSM700 High‐resolution confocal laser‐scanning microscopes (Zeiss) and Z‐stack images were obtained under a ×63 oil‐immersion objective at a definition of 1024 × 1024 pixels, adjusting the pinhole diameter to 1 Airy Unit for each emission channel. To perform ciliary length analysis, randomly chosen fields were scanned, using the same setting parameters and measurement was carried out on imaged cells (at least 50 cells/condition) for each of three‐independent experiments using the ImageJ plugin CiliaQ. Significance was determined with paired t‐testing. The percentage of ciliated cells was evaluated by counting the number of cilia in respect to the number of nuclei and was performed using the multi‐point tool of ImageJ  to facilitate the attachment of cells in 24‐well plates and cultured in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 20% FBS, 1 mM 4CENPF variants: p.Glu2178Lys  and p.Val2387Ala . BES043 fibroblasts were from a case with developmental delay but neither dysmorphic features nor microcephaly.Immunostaining of skin fibroblasts of proband 1 (NA108) for centrioles and axonemes with γ‐tubulin (red) and ARL13B (green), respectively, showed smaller cilia , we could provide functional evidence of a ciliary defect, supporting the pathogenicity of the CENPF missense variants. In contrast, another suspected case with bi‐allelic CENPF variants (BES043) with nonspecific developmental delay and without microcephaly did not show any morphological abnormalities of the cilia in fibroblasts. This simple assay appears to be a valid tool for confirming the pathogenic role of CENPF variants. However, further functional studies are needed to conclude more conclusively that missense CENPF variants result in loss of protein function. Moreover, validation of this assay in further cases is required.We report four patients with biallelic CENPF pathogenic variants. However, few cases reported with macrocephaly associated with hydrocephalus and alobar holoprosencephaly have also been reported . We speculate that this wide ranges of phenotypes might be dependent on a gene‐dosage effect with biallelic LoF variants resulting in the absence of the protein product being associated with the clinical presentations that include the full spectrum of Strømme syndrome, and missense variants allowing for some degree of residual CENPF protein function being associated with phenotypes that do not include Strømme syndrome‐related internal malformations.Pathogenic variants in genes encoding centrosomal proteins cause a variable spectrum of disorders. For example In conclusion, our case series expands the genetic and clinical spectrum of CENPF‐related disease.


The use of standard screening methods could improve the detection rate of unhealthy alcohol use in patients admitted to psychiatric acute and emergency departments. The aim of the present study was to investigate the ability of the alcohol biomarker phosphatidylethanol (PEth) to identify patients with high levels of alcohol consumption prior to admission.The data were prospectively collected at admittance to an acute psychiatric department in the period January 2016 to June 2017. A blood sample for the analysis of PEth was available from 177 patients. We compared the PEth concentrations with the Alcohol Use Disorders Identification Test (AUDIT) scores during the hospital stay, and psychiatric diagnoses at discharge.p < 0.001). PEth was above the detection limit of 0.03 μmol/L in 19% of those reporting an average daily intake of zero alcohol units per day during the last week before admission. PEth concentrations were significantly higher among those with an alcohol diagnosis than among those without such a diagnosis .A total of 45.8% of the patients had a PEth concentration ≥ 0.03 μmol/L, indicating significant alcohol consumption. AUDIT scores consistent with unhealthy alcohol use were present in 51.7%. There was a significant positive correlation between PEth concentrations and AUDIT scores (r = 0.631, PEth provides supplementary information on recent alcohol consumption in a psychiatric population and would be particularly helpful in patients unable or unwilling to give such information at admission.The online version contains supplementary material available at 10.1186/s12888-022-03934-y. Alcohol use disorder (AUD) is prevalent in patients with mental illness, and is associated with a higher risk of suicide, somatic illness and shorter life-expectancy –4. AUD iThe alcohol marker phosphatidylethanol (PEth) is produced only in the presence of ethanol in the body and is increasingly being used as a marker for alcohol intake and abstinence monitoring. Due to a detection window of up to several weeks after alcohol consumption, its application potential is broader than for other ethanol biomarkers . PEth haWe are not aware of any studies that have compared PEth and self-report assessment of alcohol use among patients admitted with acute psychiatric conditions. Hence, the aim of the present study was to investigate how PEth concentrations corresponds to AUDIT scores, self-reported alcohol intake and a diagnosis of alcohol disorder, and whether PEth can provide added value in a population of patients admitted to an acute psychiatric department.n = 177), a PEth analysis was performed. Due to the late introduction of alcohol questionnaires in the study, AUDIT was performed on a selection of patients from whom a PEth concentration was available (n = 60)  were set in joint meetings in the department . At leasAUDIT is a ten-item questionnaire with a total score from 0 to 40. It is a commonly used screening tool to asses alcohol use and to identify harmful alcohol consumption and dependence developed by the World Health Organization (WHO) , 28. As Analyses of PEth in blood was performed at the Department of Clinical Pharmacology, St Olav University Hospital. The PEth 16:0/18:1 homologue was analysed with a validated ultra-performance liquid chromatography tandem mass spectrometry (UPLC®-MSMS) method described in detail previously .There is no consensus on how to interpret a given PEth concentration, neither internationally nor in Norway . HoweverData were analysed with SPSS version 27 .Continuous data were compared by independent samples t-test or Mann-Whitney U-test, as appropriate. The Spearman rank correlation was used to assess the degree of association between two variables. ROC curves were created to evaluate the diagnostic value of PEth and AUC values with 95% confidence intervals (CIs) were calculated. Youden index (Youden’s J statistic) was calculated as J = sensitivity + specificity – 1. The index was defined for all points of the ROC curves, and its maximum value was used as a criterion for selecting optimum PEth cut-off concentrations.A PEth analysis was performed in 177 participants, and an extended AUDIT test was performed on 60 of these  had PEth concentrations ≥0.03 μmol/L, whereas 32 (18.1%) had PEth concentrations ≥0.30 μmol/L. Consequently, 96 patients (54.2%) had PEth concentrations < 0.03 μmol/L. The distribution of PEth concentrations according to gender is presented in Fig. p < 0.001). Mean PEth concentrations in those with AUDIT scores ≥8 and < 8 were 0.36 μmol/L (SD 0.63) and 0.06 μmol/L (SD 0.15), respectively (p = 0.001). Women with AUDIT scores ≥8 had a mean PEth concentration of 0.15 μmol/L (SD 0.17), while men in the same group had a mean PEth concentration of 0.58 μmol/L (SD 0.86) (p = 0.066). In the group with AUDIT scores < 8 women had a mean PEth concentration of 0.04 μmol/L (SD 0.10) and men 0.08 μmol/L (SD 0.18) (p = 0.78).Among those with available AUDIT scores, 31 (51.7%) had scores ≥8, i.e., consistent with unhealthy alcohol use  with AUDIT scores < 8 had PEth concentrations ≥0.03 μmol/L, including two (6.9%) with concentrations ≥0.30 μmol/L. The two patients having AUDIT scores < 8 and PEth concentrations ≥0.30 μmol/L, had PEth concentrations of 0.39 and 0.65 μmol/L, respectively.p < 0.001)  Fig. b. Among p < 0.001)  received a main or secondary diagnosis of alcohol disorder (F10.0 - F10.9) during their hospital stay. PEth concentrations ≥0.3 μmol/L were found in 21 (77.8%) of these. Conversely, a diagnosis of alcohol disorder was assigned to 21 of the 32 patients (65.6%) with PEth concentrations ≥0.3 μmol/L. PEth concentrations were significantly higher among those diagnosed with an alcohol disorder than among those without such a diagnosis 0.82 μmol/L (SD 0.92) vs. 0.09 μmol/L (SD 0.27); % receiveROC curves for PEth as a marker for high AUDIT score, alcohol intake and having an alcohol disorder are shown in Fig. The main finding in the present study was the significant correlation between PEth concentrations and AUDIT scores, and between PEth concentrations and self-reported alcohol consumption. Patients that received an alcohol disorder diagnosis during their stay had significant higher PEth concentrations than those who did not. More than 50% of the participants reported unhealthy alcohol use according to the AUDIT score, and almost one of five among those reporting no intake of alcohol the last week tested positive for PEth, indicating significant alcohol consumption.We decided to operate with a cut-off value for AUDIT of eight for both men and women. This is in accordance with the WHO guidelines from 2001 . Lower cFor PEth, as mentioned earlier, there are no national or international guidelines with respect to clinical interpretation of its concentrations, but the guidelines used in Norway are common practice in clinical settings in several countries . There iPEth concentrations and its clinical interpretation suggest that a consumption of two to four alcohol units several days a week is necessary to give a PEth concentration at or above 0.03 μmol/L, indicating significant or moderate consumption . Heavy cA key aim of the present study was to compare PEth concentrations with AUDIT scores . One can assume that for most participants the drinking pattern during the previous 2-4 weeks would be the same as the last year, however the fact that the two tests reflect different time periods may explain that not all participants with an AUDIT score ≥ 8 had a PEth value that indicated heavy alcohol consumption and vice versa. Different time windows may also explain why some people had a positive PEth even though they reported no alcohol intake the last week prior to admission, as questions about alcohol consumption in the preceding weeks were not included. It can be assumed that alcohol intake last week also reflects average alcohol intake in previous weeks, but for future research that compares PEth with reported consumption, it would be wise to obtain information about alcohol intake the last month.Another possible explanation for the discrepancy when comparing AUDIT scores ≥8 with PEth concentrations ≥0.30 μmol/L is that the applied cut-off for PEth may be too high. Afshar et al. recently found that a PEth cut-off of 0.04 μmol/L (25 ng/mL) was optimal for screening for unhealthy alcohol use when using AUDIT scores of ≥5 for females and ≥ 8 for males as the reference standard . Also GeNo previous studies have been performed in psychiatric emergency patients using PEth as a marker of alcohol use. However, PEth has been evaluated to identify unhealthy alcohol consumption in several populations of somatic patients. A cross-sectional study of acutely medically ill patients in Oslo, Norway, showed that the prevalence of unhealthy alcohol use was 11.4% when assessed by PEth concentrations ≥0.3 μmol/L, compared to 21.1% when using the AUDIT-4 questionnaire . In contThere are some factors that are worth noting when comparing the results from the present study with other studies. First, the study population that had completed the extended AUDIT questionnaire was small (only 60 patients included), which made it particularly difficult to evaluate subgroups. Second, in Norway, patients with symptoms primarily due to substance abuse are admitted to specialized substance abuse facilities and only patients with dominating psychiatric conditions are admitted to acute psychiatric inpatient departments. Third, also to be noted is that short hospital stays are characteristic of patients with AUD  and thatWe found a significant correlation between PEth concentrations, self-reported alcohol consumption and the presence of the diagnosis of alcohol disorder. More than 50% of patients admitted for acute mental care reported unhealthy alcohol use and 21.7% reported alcohol dependence. Such large numbers indicate that a high focus on alcohol screening is needed in acute psychiatric care to be able to give appropriate treatment. As almost one of five among those reporting no intake of alcohol the last week tested positive for PEth, a PEth analysis may provide important additional information on alcohol consumption. PEth would also be a useful tool in situations where patients are unable or unwilling to report their alcohol intake.Additional file 1.Additional file 2: Supplementary Table 1. Phosphatidylethanol (PEth) concentrations in μmol/L in the complete study population, and in women and men shown separately.


CT‐P39 is being developed as a biosimilar of reference omalizumab. This study aimed to assess the pharmacokinetic equivalence of CT‐P39 to European Union‐approved and United States‐licensed reference omalizumab .0–last), AUC from time zero to infinity (AUC0‐inf), and maximum serum concentration (Cmax). Equivalence was concluded if 90% confidence intervals (CIs) of the geometric least‐squares means ratios were contained within the predefined 80%–125% equivalence margin. Additional pharmacokinetic parameters, pharmacodynamics, safety, and immunogenicity were also evaluated.This two‐part, randomised, parallel‐group, double‐blind Phase 1 trial (NCT04018313) was conducted in healthy individuals with a total immunoglobulin E (IgE) level ≤100  international units (IU)/ml at screening. In part 2, described herein, participants were randomised (1:1:1) to receive a single 150 mg subcutaneous dose of CT‐P39, EU‐omalizumab, or US‐omalizumab. The primary endpoint was pharmacokinetic equivalence in terms of area under the concentration–time curve (AUC) from time zero to the last quantifiable concentration . For all primary pharmacokinetic parameters, 90% CIs for pairwise treatment comparisons were within the 80%–125% equivalence margin, demonstrating pharmacokinetic equivalence. Decreases in free IgE and increases in total IgE serum concentrations were comparable across groups. CT‐P39 was well tolerated. Safety endpoints were comparable across groups: there were no treatment‐related serious adverse events, deaths, or discontinuations due to treatment‐emergent adverse events.Overall, 146 participants were randomised (CT‐P39, CT‐P39 was well tolerated and demonstrated pharmacokinetic equivalence with EU‐omalizumab and US‐omalizumab following administration of a single dose in healthy individuals. A parallel‐group design was adopted to prevent potential crossover effects arising from the long half‐life of reference omalizumab (averaging 24 days in patients with chronic spontaneous urticaria and 26 days in patients with asthma).This was a two‐part, randomised, parallel‐group, double‐blind, Phase 1 trial  and provided concealed in envelopes to unblinded pharmacy personnel who dispensed study drugs in sealed cartons for administration. Randomisation was stratified by body weight (<70 kg vs. ≥70 kg), serum total IgE level , and sex .On Day 1, participants received a single dose (150 mg/1 ml) of assigned study drug via a single subcutaneous injection using a PFS into the outer upper arm. Study drugs were administered by designated clinical staff at the study centre who were not blinded to treatment assignment as PFS appearance differed between study drugs. Participants were blinded to treatment assignment through the use of a blindfold, screen, or similar method during study drug administration. Blinded staff who were not present during study drug administration performed all clinical and safety evaluations. All participants were followed up for 127 days , and PK, PD, safety, and immunogenicity were assessed.This study was conducted in accordance with the Declaration of Helsinki, the International Council for Harmonisation Guideline for Good Clinical Practice, and all applicable laws or regulations. Participants provided written informed consent before enrolment. The informed consent form and study protocol were approved by the Bellberry Human Research Ethics Committee before study initiation.2.22 (inclusive). Eligible individuals had a total IgE level ≤100 IU/ml at screening, to avoid potentially high variability in the PK profile of omalizumab. Key exclusion criteria were: current presence of clinically significant allergic disease, including asthma, urticaria, and eczematous dermatitis; history of anaphylactic shock or hypersensitivity; history of allergic reaction or sensitivity to latex or latex‐derived products; history of and/or concomitant immune complex disease (including type III hypersensitivity), hyperimmunoglobulin E syndrome, autoimmune disease, or bronchopulmonary aspergillosis; current parasitic infection or colonisation on stool evaluation for ova and parasites (assessed in participants considered to be at risk for parasitic infection); history of or any concomitant active malignancy ; history of treatment with monoclonal antibodies or other proteins targeting IgE; and pregnancy.Full inclusion and exclusion criteria are included in the Supplementary 2.30–last), area under the concentration–time curve from time zero to infinity (AUC0–inf), and maximum serum concentration (Cmax). Additional PK parameters were evaluated as secondary endpoints, including the time to maximum serum concentration (Tmax), terminal half‐life (t1/2), terminal elimination rate constant (λz), apparent total body clearance (CL/F), apparent volume of distribution during the terminal phase (Vz/F), and percentage of the AUC0–inf obtained by extrapolation (%AUCext). Secondary PD endpoints assessed for free IgE comprised the minimum serum concentration (Cmin), time to minimum serum concentration (Tmin), and maximum percentage decrease from baseline; for total IgE, they comprised Cmax, Tmax, and maximum percentage increase from baseline. Safety and immunogenicity were assessed throughout the study.The primary objective (which was assessed in part 2 of the study) was to evaluate the PK equivalence of CT‐P39 to EU‐omalizumab and US‐omalizumab, assessed in terms of area under the concentration–time curve from time zero to the last quantifiable concentration , pre‐dose on Day 1, at 6, 12, 24, 48, and 72 h after the start of study drug administration, and on Days 6, 8, 11, 15, 22, 29, 43, 57, 71, 85, 106, and 127. Validated electrochemiluminescence methods were used to measure total serum omalizumab levels and serum free IgE levels; total IgE  was measured using ImmunoCAP™ Total IgE (ThermoFisher Scientific). PK parameters were calculated based on measured total serum omalizumab concentrations by non‐compartmental methods, using Phoenix WinNonlin™, version 8.0 .Safety assessments included monitoring adverse events and prior/concomitant medications throughout. Clinical laboratory assessments  were conducted during screening and at Days −1, 3, 8, 15, 29, 57, 85, and 127. Adverse events of special interest (AESIs) were type I allergic reactions , injection‐site reactions (ISRs), type III hypersensitivity , and helminth infections . Serum samples for immunogenicity assessment were collected pre‐dose on Day 1 and at Days 15, 43, 85, and 127. Anti‐drug antibodies (ADAs) and neutralising antibodies  were assessed using electrochemiluminescence assays.2.5Log‐transformed primary endpoints were analysed by analysis of covariance at an alpha level of 0.05. Treatment was a fixed effect and the stratification criteria for randomisation were covariates. PK equivalence was confirmed for each pairwise comparison  if the 90% confidence intervals (CIs) of the ratios of geometric least‐squares means (gLSMs) were contained within the predefined equivalence margin of 80%–125%. In part 2, a sample size of 42 in each group was required to provide 90% statistical power to demonstrate PK equivalence, based on the equivalence margin, two one‐sided tests, each with an alpha level of 0.05, a 30% coefficient of variation, and an expected ratio of 1.03. Assuming a 15% dropout rate, 147 participants (49 per group) were required for enrolment.n) and percentages; continuous variables were presented using descriptive statistics. Statistical analyses were performed using SAS version 9.4.Analysis sets are described in the Supplementary 33.1N = 47; EU‐omalizumab, N = 49; US‐omalizumab, N = 50 , one was lost to follow‐up , and one withdrew due to relocating . No major protocol deviations leading to exclusion from analysis were reported during the study.The first participant was randomised to part 2 of the study on 24 June 2020; the last participant's last visit was on 27 March 2021. In part 2, 146 participants were randomised and received study drug: CT‐P39, Demographics and baseline characteristics were generally similar between treatment groups Table . Overall3.20–last, AUC0–inf, and Cmax  and syncope ; both were grade 3 in intensity and evaluated by investigators to be unrelated to the study drug. The head injury was the sole treatment‐emergent serious adverse event (TESAE) reported in this study, and the participant recovered with medical treatment. Headache was the most frequent TEAE, reported by 11 (23.4%), 10 (20.4%), and 15 (30.0%) participants in the CT‐P39, EU‐omalizumab, and US‐omalizumab groups, respectively  were reported in 33 (70.2%), 37 75.5%), and 34 (68.0%) participants in the CT‐P39, EU‐omalizumab, and US‐omalizumab groups, respectively , serum sickness/serum sickness‐like reactions, or helminth infections during the study. ISR was the only AESI reported and the second most frequently reported TEAE overall , 13 (26.5%), and 18 (36.0%) participants in the CT‐P39, EU‐omalizumab, and US‐omalizumab groups, respectively, had at least one positive ADA result. At least one positive NAb result was reported in 1 (2.0%) and 3 (6.0%) participants in the EU‐omalizumab and US‐omalizumab groups, respectively. No participants in the CT‐P39 group had detectable positive NAb titres.4Cmin, Tmin, and maximum percentage decrease. Increases in total IgE level were comparable between groups, although numerical differences were noted for the maximum percentage increase in total IgE. This was likely driven by inter‐participant variation and/or aberrant values, with one notable outlier observed in the US‐omalizumab group. The value was greatly divergent from that for other participants, but no clear factor that could account for the difference was identified. Secondary PK parameters and safety findings were generally comparable between treatment groups.Mean peak exposure and total exposure are key parameters for the demonstration of PK similarity of a candidate biosimilar to its reference product during a biosimilar clinical development programme.In the current study, the PK parameters observed across groups were broadly similar to those previously noted in a study evaluating reference omalizumab in adults with stable atopic disease.Overall, the safety profile of CT‐P39 was comparable to those of EU‐omalizumab and US‐omalizumab. In this study, the most frequently reported TEAE was headache. This is broadly aligned with the ‘common’ incidence of headache described in the EU prescribing information for reference omalizumab, which is based on clinical studies in patients with allergic asthma, chronic rhinosinusitis with nasal polyps, and chronic spontaneous urticaria.Notably, lower immunogenicity was observed in the CT‐P39 group than in the two reference omalizumab groups. However, these results should be interpreted with caution as the sample size was relatively small. In addition, no participant experienced an increase in ADA titre after treatment and the majority of ADA‐positive participants had a transiently low ADA titre of 1 in 25, the minimum required dilution of the assay. Relatively high immunogenicity was reported for reference omalizumab in this study compared with that in previous studies in patients with allergic disease,A limitation of the present study is that most (>75%) participants in each group were White, potentially limiting generalisability to other racial or ethnic groups. However, previous research with reference omalizumab has not found any clinically meaningful differences in PK and PD based on race, and efficacy and safety profiles do not differ by race or ethnicity.In conclusion, this study demonstrated PK equivalence of CT‐P39 to EU‐omalizumab and US‐omalizumab following administration of a single dose in healthy individuals. As part of a stepwise biosimilar development programme, this study provides PK evidence of high similarity for CT‐P39 and reference omalizumab. A single dose of CT‐P39 was found to be safe and well tolerated.All authors have made substantial contributions to the conception and design of the study or the acquisition, analysis, or interpretation of data; drafted the manuscript or revised it critically for important intellectual content; given final approval of the version to be published; and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.Marcus Maurer reports current or recent speaker and/or advisory roles for and/or has received research funding from Allakos, Amgen, Aralez, Argenx, AstraZeneca, Celldex, Celltrion, Centogene, CSL Behring, FAES, Genentech, GIInnovation, Innate Pharma, Kyowa Kirin, Leo Pharma, Lilly, Menarini, Moxie, Novartis, Roche, Sanofi/Regeneron, ThirdHarmonicBio, UCB, and Uriach. Sarbjit S. Saini reports research interests for Amgen, National Institutes of Health, Novartis, Regeneron, and Sanofi; and consultancies for Allakos, Aquestive, Celltrion, Escient, Granular Therapeutics, Innate, Novartis, Regeneron, and Sanofi. Sunghyun Kim, Keumyoung Ahn, Suyoung Kim, and Sewon Lee are employees of Celltrion, Inc. Clive Grattan reports consultancies for Argenx, Blueprint Medicines, Celltrion, Inc., Novartis, and Sanofi. Kristi McLendon and Paul Wabnitz have no conflicts of interest to declare.Selected results from the CT‐P39 1.1 study have been presented as posters at the Urticaria Centers of Reference and Excellence (UCARE) meeting  and the American Academy of Allergy Asthma & Immunology (AAAAI) Annual Meeting .Supporting Information S1Click here for additional data file.


Multiple myeloma (MM) results from malignant plasma cell disorder. It represents approximately 10% of hematological malignancies and it is typically diagnosed in the elderly with a median age of 70 years and has a steep increase in incidence with advancing age. N Engl J Med. 2004, 1860; Clin Interv Aging. 2020, 619. The incidence of MM has been increasing over time, mostly due to population aging. Mayo Clin Proc. 2010, 225 However, certain MMs are diagnosed at young age even under 40 years old (2%). Leuk Lymphoma. 1998, 493; Blood. 2010, 5501. We report a case of a MM in a thirty‐four‐year‐old woman whose circumstance of discovery was acute kidney failure. (A)Trichrome stain: Intratubular polychromatophilic cylinder with cellular reaction all around (×200). (B) Trichrome stain: Presence of giant cells (×400). (C) Trichrome stain: Optically normal glomeruli (×200). (D) Direct Immunofluorescence: Absence of Ig deposits but presence of lambda on tubular basement membranes and cylinders. S.W, 34, with no medical history, was admitted for investigation of advanced renal failure. On admission, she was asthenic and complained of headache and gastric pain. On examination, she weighed 62 kg, she had normal blood pressure, correct hydration, and no abnormalities on cardiopulmonary auscultations. she had a preserved diuresis (1 L). The urine dipstick showed proteinuria: + and no hematuria.At biological assessment, she had serum creatinine at 707 μmol/L, normochromic normocytic anemia at 5.2 g/dL with direct positive IgG‐type Coombs test, serum calcium at 2.8 mmol/L and LDH level at 488 mmol/L. At protein electrophoresis: monoclonal peak in gamma position. Twenty‐four‐hour proteinuria dosage was 7 g/24 H, in contrast with low proteinuria with labstix. the blood ionogram showed a natremia at 140 mmol/L, a kalemia at 5.6 mmol/L and a chloremia at 110 mmol/L. At serum immunoelectrophoretic profile, the presence of a monoclonal gammopathy type Ig G Lambda Figure . The dis2Characteristics and prognosis of young patients with MM are not well known, and available literature comes from case reports and small series.3Little is known about the disease characteristics and prognosis of these patients. The diagnosis of myeloma may result from the workup of unexplained renal disease. This diagnosis must be considered even at a young age since it involves the patient's prognosis, especially in the presence of an unexplained renal failure which should lead to renal biopsy.The first author made substantial contributions to acquisition and interpretation of data and writing the manuscript. The second author has been involved in revising it critically for important intellectual content. The third author contributed through the examination of histopathological lesions in renal biopsy and providing the photos. The last author has given final approval for the version to be published.No funding to declare.None declared.The patient has given his written informed consent to publish his case including publication of images.Written informed consent was obtained from the patient.


N = 5268 training dataset; N = 2336 test dataset)) and (b) female fish and shellfish consumers, 15–44 years of age ). Unadjusted and adjusted linear and spline models with direct measurements for both THg and MeHg were evaluated. The mean whole blood MeHg/THg ratio was 0.75 : 0.74, 0.75). This ratio was significantly higher among those with higher THg concentrations. All models exhibited excellent fit (adjusted R2 from 0.957 to 0.982). Performance was slightly improved in spline versus linear models. For the full adult population and women of reproductive age, the unadjusted spline model predicted whole blood MeHg concentrations of 5.65 µg/L and 5.55 µg/L, respectively, when the THg concentration was 5.80 µg/L. These results suggest that whole blood THg is a good predictor of whole blood MeHg among fish and shellfish consumers.Many studies evaluating methylmercury (MeHg) toxicity rely on whole blood total mercury (THg) measurements to estimate MeHg exposure. However, whole blood THg includes other forms of mercury (Hg), such as inorganic Hg, which have different exposure sources and toxicological effects than MeHg. Therefore, estimating the whole blood MeHg/THg ratio is critical to predicting MeHg exposure and, subsequently, efforts to establish an exposure–response relationship for use in risk assessment. A large, representative dataset  2011–2016) was used to determine the whole blood MeHg/THg ratio among (a) self-reported fish and shellfish consumers, ≥ 15 years of age (the “full adult” population (The online version contains supplementary material available at 10.1007/s12011-021-02968-9. Mercury (Hg) is a naturally occurring heavy metal of great concern to public health. Ample research has demonstrated that exposure to various forms of Hg is associated with impaired neurodevelopment –3, nervoThe chemical form of Hg affects its environmental fate and transport, sources of exposure, and toxicity. Health concerns have been associated with exposure to elemental Hg (Hg vapor) , 12, inoConsequently, regulatory agencies have developed guidelines specific to MeHg, including a reference dose by the US Environmental Protection Agency (US EPA) , 19. TheMore recent studies have suggested that the percentage of blood THg comprising MeHg may be highly variable as well as lower than previously estimated , with sohttps://www.cdc.gov/nchs/nhanes/index.htm.We used data from NHANES, a cross-sectional survey conducted by the US Centers for Disease Control (CDC) of the non-institutionalized civilian US population in the 50 states and the District of Columbia. NHANES consists of a questionnaire and physical exam, during which blood samples are collected. At the time of analysis, directly measured THg and MeHg concentrations were available for NHANES data collected from 2011 to 2016. These were randomly divided into two datasets: one was used to create the prediction model, i.e., the “training” dataset, and the other was used to test the model, i.e., the “test” dataset. NHANES participants sign informed consent documentation prior to participation; NHANES operates under an approved protocol from the National Center for Health Statistics (NCHS) Ethics Review Board. More details about NHANES are available online at N = 29,902 participants in NHANES 2011–2016. N = 11,953 participants were excluded because they did not have THg and/or MeHg blood measurements. Of note is that, in 2011–2012, NHANES measured THg and MeHg for the entire population providing a blood sample, but starting in 2013, only a random 50% sample of the full population who provided blood had their samples analyzed for Hg. Participants with missing data on other model covariates , N = 586; self-reported fish consumption, N = 178) were also excluded. As this analysis is heavily reliant on laboratory measurements of blood Hg, we excluded groups with a high rate of blood Hg concentrations below the detection limit: younger participants  and non-fish or shellfish consumers (31.9% of THg was < LOD). N = 5323 participants were excluded because they were less than 15 years old, and N = 2,867 participants were excluded because they did not report eating fish or shellfish within the past 30 days. This left a total of N = 7604 for analyses, referred to as the “full adult” population. In addition, as much of the existing epidemiologic studies on Hg focuses on prenatal exposures, we were specifically interested in women of reproductive age. Thus, we also created models within the subset of female fish and shellfish consumers who were of reproductive age , referred to as the “women of reproductive age” population. Two-thirds of this population was randomly selected to be in the training dataset  and the remainder were included in the test dataset .A study participant flowchart is presented in Supplemental Figure Blood samples were collected by trained phlebotomists using metal-free containers. Samples were frozen (− 30° C) until shipment to the US CDC. They were transferred to the US CDC Division of Laboratory Sciences  within the National Center for Environmental Health for Hg determination. All quality assurance and quality control protocols for Hg assessment meet the 1988 Clinical Laboratory Improvement Act mandates , 27. OthBlood THg was determined using quadrupole inductively coupled plasma mass spectrometry (ICP-MS) . This method detects the mass-to-charge ratio for each ion in the sample, which is used to determine the element and its concentration. The limit of detection (LOD) for blood THg was 0.16 µg/L in 2011–2012 and 0.28 in 2013–2016. To ensure that this did not cause any bias in our analysis, a uniform LOD (0.28) was applied to all THg samples; this affected 163/5268 measurements, or 3.1% of the full population training dataset. Values < LOD were replaced with LOD/√(2) for analyses. In the training dataset, there were 407/5268 (7.7%) values < LOD (full adult) and 121/1825 (9.4%) values < LOD (women of reproductive age). Corresponding values for the test dataset were 172/2336 (7.4%) (full adult) and 58/560 (10.4%) (women of reproductive age).MeHg concentration was determined using a triple spike isotope dilution (TSID) method; gas chromatography (GC)  was used to separate Hg forms. This was followed by inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS)  for quantification. Hg forms in blood were measured using stannous chloride as a reductant. This method has a MeHg LOD of 0.12 µg/L; this did not change over the time period included in this analysis. Similar to THg, values < LOD were replaced with LOD/√(2). In the training dataset, there were 351/5268 (6.7%) values < LOD for MeHg (full adult) and 104/1285 (8.1%) values < LOD (women of reproductive age). Corresponding values for the test dataset were 129/2336 (5.5%) (full adult) and 37/560 (6.6%) (women of reproductive age).2 and classified as normal or underweight (BMI < 25), overweight (BMI 25 to 29.9), and obese (BMI ≥ 30).Demographic data  were obtained via questionnaire. Categories used for race/ethnicity were non-Hispanic white, non-Hispanic black, Hispanic (indicated as “Mexican–American” or “Other Hispanic”), Asian, and mixed race/other. Income was defined as annual household income. Weight and height were collected at the physical examination. BMI was calculated as weight (kg) / (height (m))p value of 0.05 was considered to be statistically significant, using Pearson or Wald Chi-square tests as appropriate. The training dataset was used to create a model which would predict MeHg concentrations. NHANES is designed to obtain a representative sample of the USA; the use of appropriate survey weights and analytic methods is needed to obtain statistical results that reflect this representative sampling. However, as the goal for this analysis is not to describe the prevalence of MeHg within the US population and not all model development statistics are readily applied to survey data, survey weights and analytic methods were not used in this analysis. This could affect the variance of some variables; it also means that the population included in the analysis does not reflect a representative sample of the USA.Stata 13.0  was used for statistical analyses; a R2, mean squared error (MSE), Akaike’s Information Criterion (AIC), Bayesian Information Criterion (BIC), residuals, leverage, residual versus fitted (RVF) plots, and leverage versus squared residual (LVR2) plots. Model covariates were selected based on preliminary evaluation of their associations with blood MeHg and THg; additionally, these are commonly used covariates which have been reported to be associated with MeHg and THg in other studies. Specifically, associations of the MeHg/THg ratio have been reported with age [We explored the relationship between MeHg and THg blood concentrations. Both were approximately lognormally distributed; thus, central tendencies are reported using geometric means and log scales are used in some figures. Several unadjusted models with blood THg as the independent variable and blood MeHg as the dependent variable were created to explore model fit, including linear, linear with natural-log transformations for MeHg and/or THg, spline, and cubic spline models. We evaluated model fit using model with age , 29, sexwith age , race/etwith age , and fisUnadjusted models were created for linear, linear with natural-log transformations for MeHg and/or THg, spline, and cubic spline models. The natural-log transformed models had the poorest fit and were therefore not considered in detailed analyses (data not shown). The spline models, including the cubic spline, were roughly similar, so the best fitting spline model, with a knot at 1 µg/L THg, was used in further analyses along with the linear model.Demographic characteristics are shown in Table Average blood Hg concentrations and the MeHg/THg ratio are presented in Table Average blood Hg concentrations and the MeHg/THg ratio stratified by demographic characteristics are presented in Table β coefficient for THg was 1.01 . In unadjusted spline models among the full adult population, the β coefficient for THg values ≤ 1 µg/L was 0.88 , and β for THg > 1 µg/L was 1.01 . β coefficients for THg were not substantially affected by the population or adjustment for additional covariates; all THg coefficients were statistically significant. In adjusted models, other coefficients which were statistically significant included age (women of reproductive age only), sex (full adult only), Hispanic , Asian  and non-Hispanic black , income (women of reproductive age only), and obese . An increase in blood THg at low concentrations (≤ 1 µg/L) was associated with a significantly smaller increase in estimated blood MeHg  compared to an increase in THg at higher concentrations (> 1 µg/L) .Regression model results using the training dataset to predict blood MeHg are presented in Tables R2 values for models among the full adult population, rounded to three decimal places, were 0.982, and ranged from 0.957 to 0.959 for the women of reproductive age population. Median model residuals ranged from 0.02 µg/L (interquartile range (IQR): − 0.07, 0.12) in the unadjusted spline model for the women of reproductive age population to 0.04 µg/L  in the unadjusted linear model for the full adult population.Indicators of model fit for the training dataset are shown in Table The linear THg and spline THg models were then used to predict MeHg using the test dataset; model fit statistics are presented in Table Estimated MeHg for selected values for THg using this unadjusted spline model are presented in Table This analysis used a representative sample of the adult population of fish and shellfish consumers and a subset of women of reproductive ages from the USA to develop and test a model which would predict blood MeHg concentrations using blood THg concentrations. After testing several models, we selected the unadjusted spline model for prediction of MeHg: its performance is excellent, and due to its relative simplicity, it can be applied even when a limited amount of data from the original study is available.Although we are not aware of other models that estimate blood MeHg based on blood THg, several investigators have reported on a key parameter influencing this model: the MeHg/THg ratio. This analysis of the training dataset found that the average blood MeHg/THg ratio was 0.75 Table . This isIn this analysis, the MeHg/THg ratio was higher among those with higher THg concentrations. This positive correlation of the MeHg/THg ratio with THg concentration has also been observed in several prior studies , 29, 38.This analysis has a few limitations. First, the detection limit for THg in several NHANES cycles was high (0.28 µg/L). Due to this, some groups with lower THg exposure (children and those who do not consume fish or shellfish) were not included in this analysis. Additionally, estimates for blood MeHg for those with lower THg concentrations within our model may not be as precise as the estimates for those with higher THg exposure. Second, as we did not use survey weights in our analysis, our results cannot be assumed to reflect a representative sample of the US population. However, as the dataset was large, we feel that results are still robust.There are also several strengths of this analysis. These results are likely to be of great use to risk assessors who need to synthesize data from studies on health risks related to blood THg instead of blood MeHg. They are also consistent with the prior literature which compared blood THg to blood MeHg concentrations. Additionally, we present results specific to women of reproductive age, the demographic group commonly included in studies of the health effects of MeHg exposure.This manuscript describes the development of the first model, of which we are aware, that predicts whole blood MeHg based on whole blood THg. For studies evaluating MeHg toxicity that rely only on blood THg, this model can be used to convert blood THg concentrations to blood MeHg concentrations, and, therefore, to provide accurate estimates of exposure to MeHg.Supplementary file1 (PDF 4511 KB)Below is the link to the electronic supplementary material.


In this study we investigate whether persons with autism spectrum disorder (ASD) perceive social images differently than control participants (CON) in a graded perception task in which stimuli emerged from noise before dissipating into noise again. We presented either social stimuli (humans) or non-social stimuli . ASD were slower to recognize images during their emergence, but as fast as CON when indicating the dissipation of the image irrespective of its content. Social stimuli were recognized faster and remained discernable longer in both diagnostic groups. Thus, ASD participants show a largely intact preference for the processing of social images. An exploratory analysis of response subsets reveals subtle differences between groups that could be investigated in future studies. The question whether persons with autism spectrum disorder (ASD) experience social stimuli differently than control persons without ASD (CON) has been widely debated. In non-autistic persons there is a very stable preference for processing social stimuli , either reflected in shorter reaction times in a detection task for faces and body parts  or non-social scenes . All stimuli were created so that potential systematic differences in low-level image features between social and non-social images were reduced to a minimum. We assessed at which point in time – corresponding to different levels of noise – participants correctly recognized the scene. We expected that social stimuli would be recognized earlier than non-social stimuli when emerging from noise. We did not expect any differences between the two diagnostic groups in their mean recognition times, but the difference in recognition times between social and non-social stimuli was hypothesized to be less pronounced in persons with ASD.In a second task, images disappeared into noise  and participants had to report the point in time at which they were no longer able to recognize the image anymore. This task allowed us to rule out influences of potential differences in response times in participants with ASD and to control for potential effects of stimulus familiarity and stimulus heterogeneity. Given the high relevance of social information in everyday life, we predicted a generally more stable persistence of social images in comparison to non-social images, resulting in a later response for social stimuli. We expected that this difference in stability of image types was less pronounced in persons with ASD compared to control persons without ASD.As previous studies suggested a mediating effect of the complexity of presented stimuli  in an online study on the perceived degree of social content. Participants rated whether objects or persons were more prominent on a 5-point Likert scale (1 = object to 5 = person). The 25 highest and lowest scoring images were selected as “social”  or respectively “non-social”  and merged to hybrid images by an in-house developed algorithm . The pixels in each case were extracted in an alternating manner from two original images of the different categories. Then the extracted pixels were merged and arranged in a checkerboard order, resulting in hybrid images that combine 50% original pixels each from images of the two categories. As pixels of the initial images are merged in an alternating manner, the recognizability of one image simultaneously disturbs the recognizability of the other one. However, it is possible to control each portion separately and specifically because of the underlying regular checkerboard pattern. A systematic rearrangement of the pixel values of the one or the other source allows a controlled vanishing by stepwise randomization or respectively re-emergence of either the one or the other image. Hence, either the image from the social or the non-social category  and 23 persons with ASD  diagnosed at the outpatient clinic of the University Hospital Cologne participated in the experiment.26 persons without a diagnosis of ASD  = 2.51, p = 0.02).Participants with ASD were screened with common self-report instruments  were presented centrally on a 23″ monitor, participants were seated at approximate 60 cm distance for an image size of 7.6*7.6 degrees visual angle.Starting with a hybrid image with all pixels shuffled 100% noise), the pixels from one of the two composite images were subsequently re-arranged letting this image emerge over the course of 8 s . Subsequently, the correctly ordered pixels were shuffled incrementally over the course of 8 s, too, so that the visible image dissipated into noise see Fig. c. Duringt  and lme4 . All differences between groups were calculated using glm with Poisson link functions. We excluded trials in which no press of the space-key was recorded as missing data . To account for accidental key presses, response times below one second were excluded from analysis . Furthermore, we excluded inaccurately categorized trials from the recognition task . Finally, we excluded individual outliers, which were defined as recognition or persistence times that were above or below 2 standard deviations of the participant´s individual average in that task .(23) = 25.013, p < 0.001) and non-social stimuli  = 14.675, p < 0.001). The effect of response subsets and its interactions with other factors were tested in likelihood ratio tests of linear models. Subsequently, we also compared the recognition response times of the diagnostic groups quantile-wise for the different levels of complexity via the shift-function of the rogme package  and a “late responses” (13 stimuli) subset . The ranking was highly consistent in both groups, resulting in high correlations of average recognition times per stimulus between diagnostic groups for social  = 0.503, p = 0.50), gender (χ2(1) = 1.618, p = 0.20); ASD recognition task: age (χ2(1) = 0.505, p = 0.48), gender (χ2(1) = 0.749, p = 0.39); CON persistence task: age (χ2(1) = 0.704, p = 0.40), gender (χ2(1) = 0.023, p = 0.88); ASD persistence task: age (χ2(1) = 0.001, p = 0.98), gender χ2(1) = 2.117, p = 0.15)]. Hence, we found no support for influences of age or gender onto recognition or persistence times.To investigate possible effects of age and gender we analyzed both diagnostic groups separately. Neither in the CON group nor the ASD group any of the factors age and gender showed an increased model fit in any of the two tasks ). Social stimuli significantly lowered recognition time by 0.423 s (se = 0.07 s), (χ2(1) = 74.760, p < 0.001, dt = − 0.335 ). There was no significant interaction of group and image type (χ2(1) = 0.028, p = 0.867, dt = − 0.013 , see Fig. Belonging to group ASD significantly increased the recognition time by 0.382 s (se = 0.16 s) (χ2(1) = 2.133, p = 0.14, dt = − 0.290 ). Social stimuli increased persistence time by about 0.089 s (se = 0.044 s) (χ2(1) = 11.689, dt = 0.093 , p < 0.001). There was no significant interaction of group and image type (χ2(1) = 0.199, p = 0.66, dt = 0.028 , see Fig. Belonging to group CON did not affect persistence time (χ2(1) = 3.862, p = 0.049, dt = − 0.294 ) compared to all models including single interactions with an estimate  of 0.307 s (se = 0.16 s). In social stimuli, group differences appeared larger in early compared to late responses while in non-social stimuli, group differences appeared smaller for early compared to late responses  = 2.369, p = 0.12, subset late χ2(1) = 1.634, p = 0.20).The introduction of a three-way interaction of response subset*image type*group significantly improved the model fit  might be less heterogeneous than non-social images and are more frequently presented. This could have biased attention and could have facilitated the recognition of social stimuli. However, in the persistence task, all stimuli were already familiar and visible in the beginning of the trial, thus minimizing attention and homogeneity biases. Here, again, social stimuli seem to have been recognizable baring higher levels of noise . Thus, the persistence task further corroborates that social stimuli are indeed processed preferentially. Furthermore, although a recent meta-analysis suggests no general impairment, mixed findings exist regarding reaction times in ASD Ferraro, . TherefoResults in the persistence task did not differ between diagnostic groups and thus did not indicate any general tendency towards response delay or increased avoidance of uncertainty in ASD. We also have no reason to assume that social scenes compared to non-social scenes persisted longer in control subjects compared to subjects with ASD. One explanation could be that control participants were superior in the automatic processing of social stimuli and that this was more decisive in the recognition task. Implicit and automatic features of social cognition have repeatedly been emphasized Bargh,  and it sWe did not find specific effects of social stimuli in persons with ASD. This stands in contrast to previous studies, reporting decreased attention for social stimuli in ASD. However, recently, the account of a general reduction in attention to social stimuli in ASD has been challenged . However, separate analyses of the influence of age and gender did not point towards significant influences on the tasks. BDI scores were slightly higher in the ASD group compared to controls but none of these were above a clinically relevant threshold and the validity of these scores might be limited in persons with ASD (Cassidy et al.,


Activation of the coagulation cascade is a critical, evolutionarily conserved mechanism that maintains hemostasis by rapidly forming blood clots in response to blood‐borne infections and damaged blood vessels. Coagulation is a key component of innate immunity since it prevents bacterial dissemination and can provoke inflammation. The term immunothrombosis describes the process by which the innate immune response drives aberrant coagulation, which can result in a lethal condition termed disseminated intravascular coagulation, often seen in sepsis. In this review, we describe the recently uncovered molecular mechanisms underlying inflammasome‐ and STING‐driven immunothrombosis induced by bacterial and viral infections, culminating in tissue factor (TF) activation and release. Current anticoagulant therapeutics, while effective, are associated with a life‐threatening bleeding risk, requiring the urgent development of new treatments. Targeting immunothrombosis may provide a safer option. Thus, we highlight preclinical tools which target TF and/or block canonical (NLRP3) or noncanonical (caspase‐11) inflammasome activation as well as STING‐driven TF release and discuss clinically approved drugs which block key immunothrombotic processes and, therefore, may be redeployed as safer anticoagulants. Detection of pathogens, such as bacteria and viruses, by pattern recognition receptors (PRRs) triggers activation of components of immune cell defense pathways such as the inflammasome, type I interferon, and STING. This culminates in proinflammatory cell death, termed pyroptosis, and release of the coagulation activator, tissue factor (TF). This process of immune cell activation triggering coagulation, termed immunothrombosis, is a hallmark of coagulopathies such as sepsis and disseminated intravascular coagulation. Coagulation is a core component in maintaining physiological hemostasis and the host response to infection. The coagulation cascade is defined by two major pathways—the intrinsic and extrinsic pathways—which culminate in a common pathway which ultimately results in formation of a thrombus and fibrin clot, stopping bleeding. The intrinsic pathway, which primarily contributes to pathological clot formation , is initCoagulopathies, including sepsis and DIC, are conditions of significant microvasculature damage and multiorgan failure, and are the primary cause of death in intensive care units . ApproprTF is a 47‐kDa membrane glycoprotein and receptor and the key trigger of infection‐ and injury‐induced coagulation , 22–24. TF is modified in a process termed decryption, which occurs in‐part via changes in the lipid composition in the outer leaflet of the cell membrane , 10, 38,Following its release via pyroptotic pores, decrypted TF is expressed in the circulation on outer membrane vesicles , 41, 42 N‐terminal fragment of GSDMD is released, inserting into the cell membrane to form large oligomeric pores ) , 83, 84.d‐dimer and soluble CD14, the LPS receptor that is released by monocytes after LPS stimulation in vivo [Furthermore, HIV is associated with an increased risk of thrombosis. TF expression on the surface of monocytes is increased in humans infected with HIV . Express in vivo . TF expr in vivo , which l in vivo . HoweverSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) also drives a profound coagulopathy associated with COVID‐19, which is triggered by the key players of immunothrombosis . SARS‐CoCurrent clinically approved anticoagulant therapies, while highly effective, are associated with increased risk of bleeding because blood clotting, platelet aggregation, and fibrin cross‐linking are essential during normal hemostasis , 109. ThMight inhibition of the transcriptional processes that lead to inflammasome activation and pyroptosis be particularly attractive targets in this context? PAMP‐induced type I IFN and JAK‐STAT signalling is required for expression of ISGs such as caspase‐11. Baricitinib, ruxolitinib, and tofacitinib are clinically approved JAK inhibitors for the treatment of rheumatoid arthritis and myeloproliferative neoplasms , and thuDirectly targeting inflammasome activation is another strategy that has been shown to reduce immunothrombosis in several models. MCC950 is a highly selective inhibitor of NLRP3 , 118 anddiscussed in detail in Refs. [Recent clinical trials have also studied the effects of anti‐inflammatory therapies on thrombosis . Tin Refs. . Colchicin Refs. , 130. A in Refs. , 132, hiin Refs. , restricThe past decade has seen a flurry of research in the area of immunothrombosis. As targeting mediators of the coagulation cascade downstream of inflammasome activation and pyroptosis has not yielded any new, safer anticoagulant drugs , developThe authors declare that there is no conflict of interest associated with this manuscript.T.A.J.R. wrote the original draft. L.A.J.O'N. critically reviewed and edited the manuscript.4‐OI4‐octyl itaconatecGAMPcyclic guanosine monophosphate–adenosine monophosphatecGAScyclic GMP–AMP synthaseCKDchronic kidney diseaseCLPcaecal ligation and punctureCOVID‐19coronavirus disease 2019DAMPdanger‐associated molecular patternDICdisseminated intravascular coagulationDMFdimethyl fumarateECendothelial cellE. coliEscherichia coliGSDMDgasdermin DHMGB1high‐mobility group box protein 1HSVHerpes simplex virusIFNARIFN‐α/β receptorISGIFN‐stimulated geneITPR1inositol 1,4,5‐trisphosphate receptor type 1NETneutrophil extracellular trapNINJ1ninjurin1NLRP3NOD‐, LRR‐ and pyrin domain‐containing protein 3PARprotease activated receptorpoly(I:C)polyinosinic:polycytidylic acidPSphosphatidylserineRAGEreceptor for advanced glycation endproductsSARS‐CoV‐2severe acute respiratory syndrome coronavirus 2STINGstimulator of interferon genesS. pneumoniaeStreptococcus pneumoniaeTFtissue factorTFPItissue factor pathway inhibitorTMEM173transmembrane protein 173TRIFTIR‐domain‐containing adaptor‐inducing interferon‐β


Patients with TLE (n = 33) and age‐, sex‐matched healthy subjects (n = 36) underwent high‐resolution  structural network construction based on deterministic tracking of diffusion tensor imaging. Nodes were allocated to 68 cortical regions according to the Desikan–Killany atlas. The connectivity within regions was then used to predict surgical outcome. MRI processing, network reconstruction, and visualization of network changes were integrated into the NICARA (https://nicara.eu). Lower clustering coefficient and higher edge density were found for local connectivity within regions in patients, but were absent for the global network between regions . Local connectivity changes, in terms of the number of changed regions and the magnitude of changes, increased with disease duration. Local connectivity yielded a better surgical outcome prediction  than global connectivity. Connectivity within regions, compared to structural connectivity between brain regions, can be a more efficient biomarker for epilepsy assessment and surgery outcome prediction of medically intractable TLE.Finding clear connectome biomarkers for temporal lobe epilepsy (TLE) patients, in particular at early disease stages, remains a challenge. Currently, the whole‐brain structural connectomes are analyzed based on coarse parcellations . However, such global parcellation‐based connectomes may be unsuitable for detecting more localized changes in patients. Here, we use a high‐resolution network  to identify changes at the local level ( Local structure within regions from high‐resolution network reveals epilepsy abnormality.The extent of within‐region abnormal changes increases for long‐term epilepsy and bad surgery outcome patients.Within‐region change can be a novel network biomarker that is better than current approaches. T1‐weighted MRI scans used 1 mm isovoxel, FOV = 256 mm, TR = 2,500 ms and TE = 3.5 ms. All patients underwent a comprehensive pre‐surgical evaluation, and all had a confident diagnosis of mesial TLE based on semiological, electrophysiological and imaging investigations. Amygdalohippocampectomy and hippocampalsclerosis were confirmed histologically using standard criteria . Briefly, the pre‐processing steps included intensity normalization, skull stripping, and brain tissue segmentation. Cortical surfaces, such as the pial surface, were extracted and manually checked, edited so that its boundary did not cross the white matter surface boundary. All defects arising from this procedure were manually corrected. We down‐sampled the pial surface files with an output ratio of 0.1 using the Matlab toolbox “IsoMesh”  to get deterministic streamline tractography from eddy current‐corrected diffusion tensor images which have been processed in FSL (https://fsl.fmrib.ox.ac.uk/fsl/fslwiki/). Generalized q‐sample imaging (GQI) method with diffusion sampling length ratio of 1.25, 8‐fold orientation distribution function (ODF) tessellation with five peaks resolved on an ODF was chosen when reconstructing FIB files. Directions at corpus callosum were checked to look well . Whole‐brain seeding, 0.6*(Otsu's threshold) quantitative anisotropy level, 60 turning angle threshold, 10 mm < tracts length < 300 mm were set and a total of 10,000,000 streamlines was saved. Surface files, streamlines, and “aparc+aseg.nii” files generated by FreeSurfer were linearly registered into the same space. Streamlines whose endpoints terminating within the gray matter were acquired. The high‐resolution connectivity matrix was defined by streamline counts between the centers of the closet triangles (Euclidean distance) and was normalized by triangle area. Nodes were sorted according to the default FreeSurfer parcellation, the Desikan–Killany (DK) atlas. Each cortical DK area included hundreds of nodes and can shape an intra‐area network. We counted inter‐area streamlines and normalized with a logistic function to create weighted low‐resolution networks   , connectivity strength (S), and six topological network properties , efficiency (Ei), and clustering coefficient (Ci). Average FL was the sum of all of the streamline trajectory lengths divided by the total number of streamlines. Connectivity strength was the sum of connection weights which were normalized by triangle SA for high‐resolution networks  represents the ratio of present connections relative to the number of possible connections. Characteristic path length (L) is the average number of connections within the shortest path from one node to another. Global efficiency  is a measure of the shortest paths in the network. The average clustering coefficient (C) measures how well neighbors of a node are connected. Local efficiency  is an alternative measure of connectivity between neighbors. As the previous study mentioned, above properties are always sensitive to the sparsity of the network . Brain Connectivity Toolbox was used to compute network properties  had a negative association with the duration of epilepsy within the hippocampus, temporal lobe and several limbic structures , discriminant (DM), logistic regression (LR), support vector machines (SVM), k‐nearest neighbor (KNN), and ensemble (ENS). To avoid overfitting, 5‐fold cross‐validation was performed by partitioning the data set into folds and estimating accuracy on each fold. The same procedure was repeated and the average estimation was taken over 50 times. The optimal point of receiver operating characteristic (ROC) curve was used as a threshold for classification and was shown as the best prediction point. To explore how metrics make the good and poor surgical outcome, 5,000 permutation tests and Cohen's 33.1between‐nodes) in patients.In line with previous studies  and local efficiency  in ipsilateral PREC, specifically given that more local neighbor nodes were disconnected  in Figures within‐region networks less localized leading to lower clustering coefficient and small‐worldness (σ).To show examples for within‐region local changes, we select two regions which have the most changes in patients compared with controls . Compared to shorter duration, for longer duration more ipsilateral regions were affected beyond the temporal‐frontal regions , 93.81±0.60% accuracy, 90.38±0.67% sensitivity, and 99.83±1.18% specificity. If considering SA effect  which is similar to the 74.05±1.91% accuracy (AUC: 0.79±0.03) we get for the same region based on intra‐regional local connectivity. Moreover, across all regions, the top prediction is 0.76±0.02 AUC, 81.58±1.03% accuracy for inter‐area global network metrics . Predictive performance was better than for the standard approach of observing global changes in connections between regions indicating that within‐area connectivity can be useful in the future for planning surgical interventions.By using structural connectivity 4.1. Besides, its higher connectivity strength was a good outcome predictor , accuracy (mean: 95.39% vs. 91.45%), and specificity (mean: 100% vs. 88.67%). The predictive power of poor surgery outcome expressed by specificity in our work is crucial since alternative resection targets could be suggested instead of the planned approach. While some of the regions related to bad outcome are also regions relate to a longer disease duration, which in turn makes a bad outcome more likely , other indicated regions are independent of duration. Notably, not all changed regions are facilitating surgery and would therefore be potential surgery targets. Some changes could be due to a relocation of function away from regions affected by seizures that can no longer perform a function. For example, the developing brain can re‐establish language organization in the right hemisphere after left‐side injury . The final prediction results based on the new 360‐node network were shown in In our analysis, the DK atlas was used to assign regions to the high‐resolution network. One of the motivations to select the DK atlas is that it has relatively fewer parcellations (68 regions). The computational time therefore less, especially when multiple network metrics for all regions are analyzed. The other reason is that the smallest region had around 100 nodes for a high‐resolution parcellation with about 50,000 nodes. The smallest network size matched the number of nodes at the global scale with 68 cortical regions . For an 2); the other consisted of ~12,500 nodes . The structural connectivity comparison at different resolutions was shown in d threshold  to ~12,500 nodes (~12.6 mm2), high‐resolution networks indicate stronger performance than low‐resolution networks. The density of ~50,000 nodes which are constrained by the 68‐node ROIs (DK atlas) would be preferred as the 50 k‐network showed the best prediction and was composed of bigger regions that can benefit other network analyses, such as modularity, in the future.Overall, both parcellation scheme and node density do introduce some variability in our studies. For the low‐resolution networks, compared to the structural DK atlas, the multi‐modal atlas affects the predictive capacity of epilepsy surgery outcome slightly. While there seems to be no significant difference between the geometric‐ or biological‐informed parcellation for the low‐resolution analysis, the DK atlas with larger intra‐region networks could be better for analysis of connectivity within‐regions. Besides, varying node density from ~50,000 nodes  when planning surgical interventions  than connectivity changes between regions.In conclusion, TLE patients show characteristic changes in their structural connectivity The authors declare no competing interests.AppendixS1: Supplementary materialClick here for additional data file.