TOR1AIP1, encoding the integral nuclear membrane protein LAP1 (lamina-associated polypeptide 1) with two functional isoforms LAP1B and LAP1C, have initially been linked to muscular dystrophies with variable cardiac and neurological impairment. Furthermore, a recurrent homozygous nonsense alteration, resulting in loss of both LAP1 isoforms, was identified in seven likely related individuals affected by multisystem anomalies with progeroid-like appearance and lethality within the 1st decade of life. Here, we have identified compound heterozygosity in TOR1AIP1 affecting both LAP1 isoforms in two unrelated individuals affected by congenital bilateral hearing loss, ventricular septal defect, bilateral cataracts, mild to moderate developmental delay, microcephaly, mandibular hypoplasia, short stature, progressive muscular atrophy, joint contractures and severe chronic heart failure, with much longer survival. Cellular characterization of primary fibroblasts of one affected individual revealed absence of both LAP1B and LAP1C, constitutively low lamin A/C levels, aberrant nuclear morphology including nuclear cytoplasmic channels, and premature senescence, comparable to findings in other progeroid forms of nuclear envelopathies. We additionally observed an abnormal activation of the extracellular signal-regulated kinase 1/2 (ERK 1/2). Ectopic expression of wild-type TOR1AIP1 mitigated these cellular phenotypes, providing further evidence for the causal role of identified genetic variants. Altogether, we thus further expand the TOR1AIP1-associated phenotype by identifying individuals with biallelic loss-of-function variants who survived beyond the 1st decade of life and reveal novel molecular consequences underlying the TOR1AIP1-associated disorders.Biallelic variants in The online version of this article (10.1007/s00439-019-02105-6) contains supplementary material, which is available to authorized users. LMNA (MIM: 150330)           , encoding the integral nuclear membrane protein LAP1 (lamina-associated polypeptide 1), have been described in a family with three individuals affected by muscular dystrophy with variable cardiac involvement  and seeded 24 h prior to the beginning of the experiment.Primary human dermal fibroblast cultures were established from skin biopsies taken from the patient and three healthy donors . Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum , 1% amphotericin B solution  and 1% penicillin–streptomycin solution  under 5% CODNA samples from whole blood were isolated by standard procedures. Exome sequencing of the proband 1 was performed using an Illumina GAIIx Sequencer with the Agilent SureSelect Human All Exon 50 Mb kit, as previously described  and 1 mM DTT. Total cell protein was obtained after 30 min of centrifugation . Protein concentration was determined with PierceTM BCA protein assay kit  according to manufacturer’s instructions by reading absorbance at 562 nm with Epoch microplate spectrophotometer (BioTek). Protein volumes were adjusted to bring equal amounts on gel, mixed with BoltTM LDS sample buffer  and BoltTM sample reducing agent  according to manufacturer’s instructions. Afterwards, the samples were denaturated at 70 °C for 10 min and transferred to Bolt 4–12% Bis–Tris Plus Gel . Samples were run at 165 V for 45 min in BoltTM MOPS SDS running buffer . Proteins were transferred to the nitrocellulose membrane  at 10 V for 1 h and 15 min using Mini Blot Module  and Bolt transfer buffer  according to manufacturer’s instructions.Primary fibroblasts were grown on glass coverslips, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, 0.1% Na citrate, blocked with 5% BSA in PBS and immunostained with the respective antibodies which were diluted in 5% BSA in PBS. Images were taken using Axioplan 2 imaging microscope  and captured using the AxioVision Imaging System . Nuclear abnormalities were estimated directly by visual observation at 100× magnification, in at least 100 cells per experiment.Primary antibodies used in this study were anti-beta actin , anti-ERK1/2 , anti-phospho ERK1/2 , anti-lamin A/C , anti-lamin B1 , anti-S6 kinase , anti-phospho S6 kinase  and anti-LAP1 .Cells were seeded for 24 h and the endogenous mammalian senescence-associated β-galactosidase activity  was evaluated using the Senescence β-Galactosidase Staining Kit , according to the manufacturer’s guidelines as previously described  was used in all experiments. 70.000 cells were seeded on 6-well cell culture plates for 24 h to ensure ~ 70% confluent cultures 24 h after seeding. 4 µg of plasmid DNA was transfected for additional 24 h using the Viromer Red reagent according to manufacturer’s instructions .TOR1AIP1 (Assay ID: Hs01042342_m1), LMNA (Assay ID: Hs00153462_m1), LMNB1 (Assay ID: Hs01059203_m1) and GAPDH (Assay ID: Hs02786624_g1).Total RNA was extracted with the RNAeasy mini kit (Qiagen) from primary fibroblasts that were in all in the same passage 8, and qRT-PCR with TaqMan probes was performed as previously described . Significance was determined with two-tailed Student’s 2. The height of his mother, father and unaffected brother were 156 cm, 178 cm and 176 cm, respectively. His speech was slurred, although he had good speech understanding. Taken together, the combination of his symptoms led to the clinical diagnosis of a segmental progeroid syndrome. At the age of 32 years, following acute pneumonia, he experienced a fulminant cardiac crisis. In more detail, a cardiac decompensation with ventricular fibrillation occurred that required a short-term resuscitation including defibrillation. As a result, he developed acute renal failure with the need to start dialysis. An implantable cardioverter defibrillator (ICD) and an atrial (Demers) catheter for hemodialysis were implanted , followed by intensified anticongestive and antiarrhythmic therapy. Currently, at the age of 34 years, he still has severely reduced left ventricular function, receives dialysis for chronic renal failure, and experiences increasing difficulties in climbing stairs. Regarding diagnostic genetic analysis, chromosome analyses in blood lymphocytes did not demonstrate spontaneous chromosomal instability or hypersensitivity towards the genotoxic agent mitomycin C. Array CGH, and direct sequencing of WRN, POLD1 and LMNA all gave normal results.Individual 1 is a 34-year-old male who has since birth been regularly followed up at the University Medical Center Hamburg-Eppendorf. He is the first child of two, and his unaffected unrelated parents are of European descent. His 30-year-old brother is unaffected. Due to oligohydramnios, he was born at 34 weeks of gestation with a birth weight of 1940 g (− 0.95 SD), length of 40 cm (− 2.14 SD) and an occipitofrontal head circumference (OFC) of 31 cm (− 0.71 SD). Postnatal echocardiography revealed a ventricular septal defect (VSD) that spontaneously closed before his first birthday. In addition, hypospadias, undescended testes and congenital bilateral sensorineural hearing loss were diagnosed. Motor development was unaffected, with rolling over at 7 months and walking at 12 months. At the visit at the age of 8 years, we noted short stature and microcephaly [height 116.5 cm (− 2.43 SD) and OFC 48 cm (− 3.73 SD)]. We observed facial dysmorphisms that included long eyelashes, swinging eyebrows, telecanthus, epicanthus, anteverted nostrils, a long philtrum, and mandibular hypoplasia with dental crowding and irregular teeth. He had thin hair and developed bilateral cataracts and a mild hepatosplenomegaly. Further, a delay in speech development was noted, which was at that time regarded as a consequence of bilateral hearing loss. Extensive metabolic studies, bone age assessment and brain CT yielded normal results. EEG revealed a mild increase in cerebral excitability. Conventional chromosome analysis in lymphocytes gave normal results. Since his late 20s, he has had repeatedly elevated non-fasting glucose levels ranging from 127 to 201 mg/dl (reference 60–110 mg/dl), suggestive of type 2 diabetes; however, due to the lack of compliance, confirmatory tests have not been performed. In addition, he has had repeatedly elevated blood levels of non-fasting cholesterol ranging from 205 to 220 mg/dl (reference 150–200 mg/dl), LDH ranging from 320 to 480 U/l (reference 87–241 U/l), GGT ranging from 72 to 150 U/l (reference < 65 U/l), ALT ranging from 54 to 68 U/l (reference 10–50 U/l), and creatinine ranging from 1.64 to 3.60 mg/dl (reference 0.6–1.2 mg/dl). At the visit at the age of 31 years, he presented with bilateral hearing loss, mandibular hypoplasia with dental crowding and irregular teeth, scleroderma-like skin changes, Raynaud’s phenomenon, hand joint contractures, mild intellectual disability, muscular atrophy and proximal muscle weakness, dilated cardiomyopathy and chronic heart failure with significant reduction of left ventricular ejection fraction of 35%  . His weight was 40 kg and height 157 cm, resulting in a BMI of 16.23 kg/mGJB2, GJB6, SLC52A3, SLC52A2, and SLC52A1.Individual 2 was a 16-year-old white male born after a full-term uncomplicated pregnancy to a 34-year-old G3P3 mother with birth weight of 3700 g. Both parents were unaffected and unrelated. His two siblings, age 17 and 15, respectively, were also healthy. A profound congenital sensorineural hearing loss was found at newborn screening. At 1 week of age, VSD was diagnosed, as well as an asymptomatic bicuspid pulmonary valve and persistence of a dilated pulmonary artery. The VSD was eventually closed. At 1.5 months of age, he developed bilateral cataracts and underwent surgical lens implants. He had G-tube insertion in early childhood due to feeding difficulty and failure to thrive. At his 4.5-year-old visit, he was noted to have microcephaly and short stature [OFC 47 cm (− 2.30 SD) and height 99 cm (− 1.74 SD)]. Other dysmorphic features included low-set and posteriorly rotated ears, deep-set eyes, thin nose, high-arched palate, long philtrum, thin upper lip, down-turned mouth, mandibular hypoplasia, and dental crowding. His development was globally delayed; he walked at 2.5 years, was nonverbal, but had learned 100–200 signs. There was no evidence of deterioration until he was around 11 years of age. At age 12, he developed bilateral ptosis, horizontal nystagmus, swallowing dysfunction, hypotonia, muscle weakness, gait changes, asymmetric and small thoracic size, mixed pectus carinatum and pectus excavatum, kyphoscoliosis, femoral anteversion, and cardiac arrhythmia. He had G-tube re-insertion due to difficulty swallowing. He also had surgery for bilateral ptosis and dental abnormalities. His symptoms continued to progress and at 15 years he was diagnosed with dilated cardiomyopathy with moderate left ventricular dysfunction. His left ventricular ejection fraction was 35.1%  and frequent (2.2% of time) ventricular ectopic activity was detected through a 24-h Holter electrocardiogram . At a clinical visit at age 16, he had chronic heart failure with ejection fraction only at 39%. He was mostly wheelchair-bound with global hypotonia and muscle weakness. Joint contractures were observed in his elbows, wrists, fingers and lower extremities. He also had nearly constant choreoathetoid movements and dystonic posture. His height was 149.8 cm (− 3.58 SD), weight 30.6 kg (− 5.17 SD) and head circumference 50 cm (− 4.27 SD). He died at age 16 years due to progressive heart failure. Quadriceps muscle biopsy at 1.5 years of age showed variable myofiber size with occasional small atrophic myofibers and centrally positioned nuclei; these findings were not considered specific for any specific disease processes. At the age of 12 years, brain MRI showed minimal periventricular signal abnormality but was otherwise within normal limits. CPK level was slightly decreased at 45  and lactic acid was slight elevated at 22.6 . Extensive metabolic and biochemical workup was unrevealing, including plasma amino acids, acylcarnitine, carnitine, GALT, urine amino acids, organic acids, MPS type 1, GAG, lysosomal enzyme, carbohydrate deficient transferrin, N-glycan, O-glycan, 7-dehydrocholesterol, and very-long-chain fatty acids. Negative genetic testing included FISH, karyotype, mitochondrial DNA genome sequence, cytogenomic microarray, and direct sequencing of Taken together, both individuals presented with congenital bilateral hearing loss and ventricular septal defect, followed by development of bilateral cataracts, mild to moderate developmental delay, microcephaly, mandibular hypoplasia with dental crowding and irregular teeth, relative short stature, progressive muscular atrophy, joint contractures, dilated cardiomyopathy, and reduced left ventricular ejection fraction followed by severe chronic heart failure Fig. , 2005.FiGMPPA and TOR1AIP1 (Table S2). Segregation analysis revealed that both heterozygous variants affecting GMPPA were inherited from the unaffected father, thus excluding a causative role. On the contrary, we confirmed a paternally inherited c.945_948delCAGT, p.(Gln315Hisfs*9), and a maternally inherited c.1331G > C, p.(Arg444Pro), mutation in TOR1AIP1 (NM_001267578.1)  was performed with DNA samples of both affected individuals. Bioinformatic filtering of WES data in individual 1 for putatively de novo variants detected 42 heterozygous alterations that have previously not been annotated in dbSNP, the ExAC database or the 1000 Genomes data; however, subsequent Sanger sequencing with DNA samples of both healthy parents and individual 1 revealed that each of these variants was inherited (Table S1). Further, we did not detect rare candidate variants  following an X-linked recessive mode of inheritance or any putatively pathogenic homozygous alterations. However, we identified two genes with rare  double heterozygous variants affecting TOR1AIP1, namely, a maternally inherited variant c.724delG, p.(Asp242Ilefs*17) and a second heterozygous variant c.649G > T, p.(Glu217*). As paternal DNA was not available for sequencing, we could only infer that this second variant is likely to have been inherited from his father, as it is absent in both his mother and unaffected sister. His unaffected sister is a carrier of the c.724delG, p.(Asp242Ilefs*17) , these frameshift and nonsense variants are predicted to lead to a premature termination codon, likely activating nonsense-mediated mRNA decay as was recently shown for the homozygous p.(Arg321*). According to the gnomAD database, all four identified variants are exceedingly rare: p.(Glu217*) was observed 3 times, p.(Asp242Ilefs*17) was observed 1 time, p.(Arg444Pro) was observed 16 times, whereas p.(Gln315Hisfs*9) was not annotated in this database. Taken together, the genetic data provided strong support for the causality of these TOR1AIP1 variants.Bioinformatic filtering of WES data in individual 2 also revealed two likely pathogenic variants in TOR1AIP1 variants identified in individual 1, we established primary dermal fibroblasts of individual 1. We additionally established primary dermal fibroblasts of three unaffected individuals of similar age . Individual 2 unfortunately deceased prior to skin biopsy, thus we were unable to analyze his primary cell lines. It is, however, worth noting that both of the variants identified in individual 2 lead to premature stop codons which are more N-terminal than the p.(Arg321*), for which a nonsense-mediated mRNA decay was recently shown  of individual 1 fibroblasts and fibroblasts from an unaffected control individual. We observed, relative to fibroblasts from the healthy control individual, lower levels of lamin B1 Fig. , we addivel Fig. e, g. ThuTOR1AIP1-associated disorder . Using polyethylenimine polyplex transfection technology (Viromer RED), we achieved a transfection efficiency of around 20% in passage 8 of individual 1’s fibroblasts, as revealed by immunofluorescence analysis with anti-FLAG . Immunoblotting analyses revealed that ectopic expression of TOR1AIP1 resulted in robust stabilization of lamin A/C levels as well as a partial reduction in both pS6k, as a marker of premature senescence, and pERK1/2 levels , and we observed no nuclei lacking lamin A/C staining Below is the link to the electronic supplementary material.


Soil denitrification rates were significantly higher (p < 0.05) in animal colonies than in animal-lacking tundra, and they were significantly positively correlated (p < 0.05) with nirS gene abundances instead of nirK gene abundances, indicating that nirS-encoding denitrifiers dominated the denitrification in tundra soils. The diversity of nirS-encoding denitrifiers was higher in animal colonies than in animal-lacking tundra, but the diversity of nirK-encoding denitrifiers was lower. Both the compositions of nirS‐ and nirK-encoding denitrifiers were similar in penguin or seal colony soils. Canonical correspondence analysis indicated that the community structures of nirS‐ and nirK-encoding denitrifiers were closely related to tundra soil biogeochemical processes associated with penguin or seal activities: the supply of nitrate and ammonium from penguin guano or seal excreta, and low C:N ratios. In addition, the animal activity-induced vegetation presence or absence had an important effect on tundra soil denitrifier activities and nirK-encoding denitrifier diversities. This study significantly enhanced our understanding of the compositions and dynamics of denitrifier community in tundra ecosystems of maritime Antarctica.In maritime Antarctica, sea animals, such as penguins or seals, provide a large amount of external nitrogen input into tundra soils, which greatly impact nitrogen cycle in tundra ecosystems. Denitrification, which is closely related with the denitrifiers, is a key step in nitrogen cycle. However, effects of sea animal activities on tundra soil denitrification and denitrifier community structures still have received little attention. Here, the abundance, activity, and diversity of nirS‐ and nirK-encoding denitrifiers were investigated in penguin and seal colonies, and animal-lacking tundra in maritime Antarctica. Sea animal activities increased the abundances of nirS and nirK genes, and the abundances of nirS genes were significantly higher than those of nirK genes ( Nitrogen (N) is an essential element for the biosynthesis of key cellular components, such as proteins and nucleic acids, in all organisms . Nitroge3−, NO2−, and NH4+) (Nitrite reductases (Nir) is the rate-limiting enzyme among the enzymes that catalyze the denitrification processes . Nir encnd NH4+) . At presnd NH4+) , Antarctnd NH4+) , and thend NH4+) , and micnd NH4+) . However3 or NH4+, which is subsequently oxidized to nitrate through nitrification, and eventually converted to N2 through denitrification (2O), as a strong greenhouse gas and stratospheric ozone depletion substance, can be produced during the denitrification in soils  and high-throughput sequencing were conducted to investigate the abundance and diversity of nirS‐ and nirK-encoding denitrifiers in different tundra soils. Our main objectives were (1) to examine potential denitrification rates in tundra soils; (2) to investigate the abundance, diversity, and community structures of nirS‐ and nirK-encoding denitrifiers; and (3) to determine effects of sea animal activities and environmental variables on the abundances, community compositions, and activities of the denitrifiers in tundra soils of maritime Antarctica.2. It is one of the warmest and humidest areas in Western Antarctica due to effects of sub-Antarctic maritime climate. According to the long-term meteorological data collected at Great Wall Station on this peninsula, the mean annual temperature is about −2.5°C with a range of −26.6 ~ 11.7°C, and mean annual precipitation is 630 mm with the main form of snowfall.Mirounga leonine), Weddell seal (Leptonychotes weddellii), fur seal , and leopard seal (Hydrurga leptonyx)  and Ardley Island  in the southwest of King George Island . Fildes eptonyx) . During eptonyx) .via a sand dam. This island is an important ecological reserve for penguins in Western Antarctica. The local prevailing wind direction is from west or northwest, which leads to the accumulation of less snow in the east of this island , and allows the establishment of active penguin colonies mainly in the eastern coast. There are approximately 5,100 breeding pairs including Gentoo (Pygoscelis papua), Adélie (Pygoscelis adeliae), and Chinstrap penguins (Phyllophora antarctica) in the austral summer . After collection, each soil sample was divided into two parts: one part was stored at −80°C for microbial molecular analysis, and the other part was stored at −20°C for the analyses of soil physicochemical properties and denitrification activity.4+-N) and nitrate (NO3−-N) were extracted from soils with 2 mol L−1 KCl, and measured using a continuous-flow nutrient analyzer   was measured using Inductively Coupled Plasma Optical Emission Spectrometer  (Soil samples were dried at 105°C to a constant weight to measure moisture content expressed as the percentage of weight lost. Organic matter (OM) was determined through the loss of ignition protocol, where soils were ignited in a muffle furnace for 4 h at 550°C after initial oven drying at 105°C. Soil pH was measured by mixing soil and Milli-Q water (1:2.5 ratio). Total nitrogen (TN), total carbon (TC), and total sulfur (TS) were measured using an elemental analyzer  . The ammerlands) . After d States) .15NO3−. The final concentration of 15N in each vial was approximately 100 μmol L−1. Half of the vials were immediately injected 200 μl 50% ZnCl2 solution into each vial to block slurry incubation. The remaining vials were incubated for 8 h at 10°C, and then were blocked by injecting 200 μl 50% ZnCl2 solution. The concentrations of 29N2 and 30N2 produced during the incubation were measured by membrane inlet mass spectrometry. The calculation of denitrification rates and more detailed information on slurry experiments have been described in the references  under summer collection conditions. After pre-incubated, the vials were spiked with helium-purged solutions of ferences .Taq PCR Master Mix, 1 μl of template DNA, 2 μl of each primer (10 μM) and 20 μl of Nuclease-free ddH2O up to 50 μl . The PCR condition was 2 min at 94°C, followed by 35 cycles of 94°C (30 s), 57°C (30 s), and 72°C (1 min) and a final step of 72°C (10 min) for both the nirS and nirK genes. The amplification products were sequenced using Illumina MiSeq in Sangon Company . Although primers Cd3aF:R3cdR and FlaCu:R3Cu set are not comprehensive enough to target all nirS-encoding and nirK-encoding denitrifiers, they can be used to compare the denitrifier diversity between soil samples . The extracted DNA was eluted in 60 μl of elution buffer. DNA integrity was checked by agarose gel electrophoresis . The genomic DNA was quantified using Qubit2.0 DNA Assay Kit . The nirS gene fragment (425 bp) was amplified using primers Cd3aF (5'-GTSAACGTSAAGGARACSGG-3') and R3cdR (5'-GASTTCGGRTGSGTCTTGA-3') . The nir samples .R2 = 0.9994 for nirS; R2 = 0.9992 for nirK). The melt curve was checked, and the amplification efficiencies for nirS and nirK were 90.3 and 107.6%, respectively. The standard curves were used to calculate the abundance of nirS and nirK in tundra soils.The abundances of nirS and nirK genes were determined in triplicate by qPCR using a LightCycler480 II Real-time PCR System . The primers were the same as those used in high-throughput sequencing. The standard curves showed strong correlations between the threshold cycle (Ct) and the lg values of gene copy numbers  2 Version:2019.07 . QIIME 2ue <1e−6 . NeighboH), Simpson index (1/D), and Pielou’s evenness, and the abundance-based coverage estimate Ace were calculated by the R Version:3.6.1 (H) and Simpson index (1/D) indicated alpha diversity for nirS‐ or nirK-encoding denitrifiers, and Pielou’s eveness represented the evenness of the denitrifier community in tundra soils. The abundance-based coverage was used to estimate the gene library coverage. One-way analysis of variation (ANOVA) and T-test were calculated for the comparisons between nirS, nirK gene abundances, diversity, and denitrification rates between tundra soils using SPSS Version:20. Correlations between nirS and nirK gene abundances, denitrification rates, and environmental factors were obtained by Pearson correlation analysis . Compared with animal-lacking tundra soils, active penguin and seal colony soils had much higher TN, TS, TP, and NH4+-N contents most likely due to the deposition of penguin guano or seal excreta. Especially, the NH4+-N and TS contents in penguin colony soils (means: 73.4 mg NH4+-N kg−1 and 4.3 mg S g−1) and seal colony soils (means: 62.0 mg NH4+-N kg−1 and 2.1 mg S g−1) were one to two orders of magnitude higher than those in animal-lacking tundra soils (means: 4.2 mg NH4+-N kg−1 and 0.5 mg S g−1). In addition, penguin colony soils (PS1–PS3) and its adjacent tundra soils AL1 had higher NO3−-N (1.0–12.0 mg kg−1) than seal colony soils (SS1–SS3) and animal-lackingtundra soils AL2 and AL3 (0.1–0.2 mg kg−1). Seal colony soils had lower C:N ratios (mean:5.2) than penguin colony soils (mean:8.6) and animal-lacking tundra soils (mean:11.5). Overall, the deposition of penguin guano or seal excreta altered local soil biogeochemical properties, leading to generally low C:N ratios and high TN, TS, TP, and NH4+-N contents in fauna-related tundra soils.Soil physicochemical properties showed high heterogeneity across different types of tundra sites in maritime Antarctica . Penguint-test, n = 9, p < 0.05). The abundances of nirS genes in penguin and seal colony soils (3.5 × 106–2.5 × 107 copies g−1) were two to four orders of magnitude higher than those in the animal-lacking tundra soils (5.9 × 103–4.9 × 105 copies g−1). The highest nirS gene abundance was detected in SS2, whereas the lowest occurred in AL3 (5 copies g−1) and PS3 (3.2 × 105 copies g−1), one to three orders of magnitude higher than those in other soils within animal colonies (4.5 × 104–9.1 × 104 copies g−1) and in adjacent animal-lacking tundra soils (7.4 × 102–1.5 × 104 copies g−1). The lg values of both nirS and nirK genes abundances showed significant negative correlations  with C:N ratios in tundra soils  than those in animal-lacking tundra soils. The mean highest rate occurred in seal colony soils (mean 29.88 ± 22.77 μmol N kg−1 h−1), followed by penguin colony soils (mean 11.10 ± 8.34 μmol 11 N kg−1 h−1), two orders of magnitude higher than those in animal-lacking tundra soils (mean 0.62 ± 0.50 μmol N kg−1 h−1). Of all tundra soils, the highest rate occurred at SS2 (59.49 ± 3.3 μmol N kg−1 h−1) with the highest nirS gene abundance. Even within penguin or seal colony, the denitrification rates also showed high variability between tundra soil samples due to effects of these animal activities and the deposition of their excreta. The denitrification rates in SS2, SS3, and PS3 (20.29–59.49 μmol N kg−1 h−1) were one order of magnitude higher than those in SS1, PS1, and PS2 (5.79–9.85 μmol N kg−1 h−1). The denitrification rates were significantly positively correlated with nirS gene abundances  in tundra soils, but no correlation was obtained between denitrification rates and nirK gene abundances  indexes of nirK-encoding denitrifiers were higher than those of nirS-encoding denitrifiers in animal-lacking tundra soils  but lower in sea animal colony soils. The three indexes for the nirS-encoding denitrifiers were higher in penguin or seal colony soils than in animal-lacking tundra soils (p < 0.05) from those in animal-lacking tundra soils. The Simpson (1/D) indexes for the nirK-encoding denitrifiers were significantly lower in penguin colony soils than in animal-lacking tundra soils . For the same soil sample types, the Pielou’s Evenness Index and Shannon-Wiener Index (H) for the nirS‐ and nirK-encoding denitrifiers showed a low variability with the small standard deviations of the means. However, the Simpson Index (1/D) showed a high variability even between the same soil sample types , whereas the evenness index of nirK-encoding denitrifiers showed significant negative relationships with TN, TC, and TS  was higher than that of nirS-encoding denitrifiers (128–304) . The Piera soils . On the le types . In addi, and TS .Proteobacteria in tundra soils. The most abundant 50 OTUs were grouped into distinctly defined seven clusters (I–VII) on the basis of evolutionary distance. The cluster I contained eight OTUs, and 31,340 nirS gene sequences with 53.3% of the sequences from AL, 36.5% from PS, and 10.2% from SS, and they were closely related to those in sediments of Qinghai-Tibetan Plateau river (MH634647) and Tibetan Plateau wetland (KC468828). The cluster II contained eight OTUs, and 51,450 sequences with 45.8% of the sequences from AL, 35.6% from PS, and 18.6% from SS, and they closely matched to those in estuarine sediments from San Francisco Bay (GQ453804) , accounting for 36.8% of the total 50 OTU sequences, with 43.9% from AL, 29.7% PS, and 26.4% from SS, and they closely matched to those found in Baltic Sea (EF615460.1), Yangtze lakes (KU159637), and Pearl River (JN016582). Overall, tundra soils contained all the seven clusters except AL1 and AL3. The clusters III, IV, VI, and VII appeared in all tundra soils. The dominant clusters in nirS genes from penguin and seal colony soils were II, IV, VI, and VII, accounting for 58.3–92.0% of nirS gene sequences (Pseudomonas stutzeri SLG510A3-8 (CP011854) and had 100.0% identity from the Genbank.Phylogenetic analysis of nirS genes showed that a large proportion of unique OTUs matched to uncultured environmental nirS assemblages . The majQ453804) . The cluQ453804) . The cluequences . The domMesorhizobium sp. (AY078254) (Nitrosospira (CP021106) (Desulfosarcina (AP021879), respectively. Overall, the clusters II, III, and IV were found in all the nine tundra soils. The dominant clusters from penguin and seal colony soils were I, III, and V, accounting for 62.0 to 99.7% of total sequences  on the basis of evolutionary distance . The mosY078254)  and simiY078254) . The cluY078254) , San FraY078254) , Yellow Y078254) , Lake BaY078254) , and micY078254) . The cluP021106)  and Desuequences . Cluster3−-N  and NH4+-N , which together explained 53.6% of the variation (p > 0.05), these variables additionally explained 40.7% of the variation. The first two dimensions explained 73.3% of the cumulative variation of the nirK-encoding denitrifying community-environment relationship. The nirK community structure significantly correlated with C:N  and NH4+-N , which together explained 51.3% of the variation. Other environmental variables, including NO3−-N, pH, and TC, accounted for 43.8% of the variation.The relationships of the nirS‐ and nirK-encoding denitrifier community structure with environmental variables were analyzed by the CCA. The environmental variables in the first two CCA dimensions (CCA1 and CCA2) provided 67.7% of cumulative variation of the nirS-encoding denitrifying community-environment relationship . The nirariation . Althoug6–2.5 × 107 copies g−1) in penguin and seal colony soils were close to those from the soils of King Sejong Station in Antarctica (7–108 copies g−1 soil), higher than those in Tibet Plateau soils (2.03 × 104–1.28 × 106 copies g−1) (8–1.8 × 109 copies g−1) (2–6.2 × 105 copies g−1) were significantly lower compared with nirS gene, consistent with those (103–106 copies g−1 soil) in soils from King Sejong Station in Antarcitc (9–1.00 × 1010 gene copies g−1) in penguin and seal colony soils, whereas the proportion of nirK gene abundances in the bacterial abundances were two to three orders of magnitude lower than that of nirS gene abundances  , but lowies g−1) . The nirntarcitc , and Svantarcitc . In addintarcitc  and highntarcitc . The nirundances .Our previous studies showed that penguin or seal activities led to generally low C:N ratios, which had been used as an indicator for the intensity of penguin or seal activities in maritime Antarctica . The abu−1 h−1) were significantly higher than those in adjacent animal-lacking tundra soils (0.04–0.93 μmol N kg−1 h−1) (−1 h−1) (−1 h−1) (−1 h−1) (−1 h−1) (−1 h−1)  , similarg−1 h−1) , indicat(−1 h−1) , but sig(−1 h−1) , similar(−1 h−1) , northea(−1 h−1) , and Rus(−1 h−1)  although(−1 h−1) . The den(−1 h−1) , but muc(−1 h−1) , northea(−1 h−1) , Russian(−1 h−1) , and Arc(−1 h−1) .r = 0.733, p = 0.02). However, no significant statistical correlation was obtained between the denitrification rates and nirK gene abundances, tundra soil physicochemical properties. Therefore, compared to nirK gene abundances and soil physicochemical properties, the abundances of nirS genes better predicted the denitrifier activities in tundra soils of maritime Antarctica. In this study, the animal-lacking tundra areas were covered with the cushions of mosses and lichens due to moderate amount of nutrients and the absence of animal trampling. These tundra plants can absorb the limited soil N, highly limit the inorganic N (NH4+-N) availability for the denitrifiers  showed a low variability within the same sample types (D) showed a heterogeneous distribution pattern, possibly because the inverse of Simpson Index (1/D) is sensitive to the level of dominance in a community  between Pielou’s evenness of nirS-encoding denitrifiers and tundra soil C:N ratios  and Simpson Index (1/D) for the denitrifiers and soil environmental variables, which could be due to the low number of soil sample replication within the same tundra types.For nirK-encoding denitrifiers, their diversity was lower than nirS-encoding denitrifiers in sea animal colonies, which was in accordance with other environments, such as sediments of San Francisco Bay , YangtzeProteobacteria (mean 48.6%), Actinobacteria (mean 16.3%), and Bacteroidetes (mean 8.6%), and Proteobacteria was the most dominant bacterial phylum  than in non-animal tundra soils. Therefore, the NO3− from ammonium nitrification was likely important substrate for soil denitrification in sea animal colony  can be found in the article/R-BZ, H-TD, and B-WS designed the experiments. L-JH provided experiments platform. H-TD, B-WS, and C-SC performed the experiments and analyzed the data. H-TD and R-BZ prepared original draft. R-BZ and H-TD reviewed and edited the manuscript. All authors contributed to the manuscript and gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human palatine tonsils are potential tissue source of multipotent mesenchymal stem cells (MSCs). The proliferation rate of palatine tonsil-derived MSCs (TMSCs) is far higher than that of bone marrow-derived MSCs (BMSCs) or adipose tissue-derived MSCs (ADSCs). In our previous study, we had found through DNA microarray analysis that tensin-3 (TNS3), a type of focal adhesion protein, was more highly expressed in TMSCs than in both BMSCs and ADSCs. Here, the role of TNS3 in TMSCs and its relationship with integrin were investigated. TNS3 expression was significantly elevated in TMSCs than in other cell types. Cell growth curves revealed a significant decrease in the proliferation and migration of TMSCs treated with siRNA for TNS3 (siTNS3). siTNS3 treatment upregulated p16 and p21 levels and downregulated SOX2 expression and focal adhesion kinase, protein kinase B, and c-Jun N-terminal kinase phosphorylation. siTNS3 transfection significantly reduced adipogenic differentiation of TMSCs and slightly decreased osteogenic and chondrogenic differentiation. Furthermore, TNS3 inhibition reduced active integrin beta-1 (ITGβ1) expression, while total ITGβ1 expression was not affected. Inhibition of ITGβ1 expression in TMSCs by siRNA showed similar results observed in TNS3 inhibition. Thus, TNS3 may play an important role in TMSC proliferation and differentiation by regulating active ITGβ1 expression. Recently, human palatine tonsils have emerged as a new source of mesenchymal stem cells (MSCs) ,2,3,4. PIn another previous study , we compTensin is a focal adhesion protein, of which TNS3 is one of the four tensin isoforms ,7,8. Focg for 10 min. After centrifugation, the pellet was filtered through a 100-μm nylon mesh to remove cellular debris, and the filtrate was incubated overnight in control medium  at 37 °C under a 5% CO2 atmosphere. Following incubation, the plates were washed extensively with PBS to remove residual non-adherent cells, and the resulting cell populations were further maintained. All assays, including TNS3 blocking studies, were repeated three to four times in all four TMSCs. In this study, we utilized the cells that we verified the characteristics of mesenchymal stem cells by determining the proliferation, differentiation, and surface markers, as we previously reported [The palatine tonsils were obtained from four different patients diagnosed with chronic tonsillitis who had undergone a tonsillectomy. To isolate the stem cells, the tonsils were washed extensively with equal volumes of phosphate-buffered saline (PBS), and the tissue was digested in 0.075% collagenase type I  at 37 °C for 30 min. Enzyme activity was neutralized with alpha-modified Eagle’s medium (α-MEM) containing 10% fetal bovine serum (FBS), and the sample was centrifuged at 1200× reported ,5.g for 10 min, and the pellet was incubated overnight in the control medium at 37 °C under 5% CO2. Following incubation, the tissue culture plates were washed to remove any residual non-adherent cells and then maintained in control medium at 37 °C under 5% CO2. Bone marrow samples were obtained from four volunteers. Mononuclear cells from the bone marrow were separated by centrifugation in a Ficoll–Hypaque gradient  and suspended in α-MEM containing 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. The cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2. The adherent cell monolayer at 90% confluence was trypsinized , and the cells were resuspended in α-MEM containing 10% FBS and subcultured at a concentration of 2000 cells/cm2. Cells between the third and fourth passages were used in all further experiments.ADSCs and BMSCs were isolated and characterized, as described in our previous studies ,10. The The study protocol was reviewed and approved by the Pusan National University Hospital Institutional Review Board.3 cells/cm2 with Dulbecco’s modified Eagle’s medium–low glucose containing 10% MSC-qualified FBS and incubated in a 37 °C incubator under 5% CO2. The cells were subcultured every 4–5 days to reach P28.Adherent primary TMSCs were expanded in culture, and colonies started to form after 5–6 days of isolation. The medium was replenished twice weekly. When cells reached 80–90% confluency, they were detached with a 0.25% trypsin/EDTA solution . Population doubling and cell viability were measured. Next, the cells were seeded into culture flasks at a density of 1.5 × 10PPARγ), lipoprotein lipase (LPL), osteocalcin, alkaline phosphatase (ALP), collagen type II alpha-1 (COL2A1), and GAPDH as the internal control. Primer sequences are shown in Quantitative reverse transcription (RT)-polymerase chain reaction (PCR) was performed to determine the expression levels of TNS3, SOX2, Oct-4, Nanog, c-Myc, p16, p19, p21, CDC25, cyclin E, peroxisome proliferator-activated receptor-gamma (The cells were washed twice in cold PBS and lysed in RIPA buffer (Sigma-Aldrich). Proteins were loaded into a 10% sodium dodecyl sulfate-polyacrylamide gel, electrotransferred to nitrocellulose membranes , and probed with monoclonal antibodies. Immunoreactive bands were detected using anti-mouse and anti-rabbit peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech) and visualized by enhanced chemiluminescence .Small interfering RNA (siRNA) oligonucleotide duplexes  targeting TNS3 and ITGβ1 mRNAs, and a nontargeted oligonucleotide duplex  as a negative control, were used in the transfection experiments with DharmaFECT Transfection Reagent. 4 cells/well in 24-well plates and evaluated after 1, 2, 3, and 4 days of culture. After washing the cells, the culture medium containing 0.5 mg/mL of MTT was added to each well. The cells were incubated for 2 h at 37 °C, after which the supernatant was removed, and formazan crystals that had formed in viable cells were solubilized with 200 μL of dimethyl sulfoxide. Next, a 100-μL aliquot of each sample was transferred into 96-well plates, and the absorbance of each well was measured at 560 nm using an ELISA reader . This experiment was repeated four times. Cell cycle analysis was performed using 6-well plates seeded with 5 × 104 cells/well. These cells were treated with recombinant human FGF5 for 48 h at 37 °C and harvested using 0.05% trypsin solution. Thereafter, they were centrifuged at 10,000× g for 15 min, and the pellets were washed twice in Hank’s Balanced Salt Solution buffer and fixed with 70% ethanol at −20 °C overnight. On the following day, ethanol was removed, and the cells were resuspended in 500 mL of PBS containing 1 mg/mL of propidium iodide and 100 μg of RNase/mL for 20 min, followed by analysis with a FACS Calibur .Cell proliferation was evaluated by the 3--2,5-diphenyltetrazolium bromide (MTT) assay and cell cycle analysis. For the MTT assay, TMSCs were plated at a density of 1 × 105) were plated into the upper chamber, while the lower chamber was filled with media containing interferon-γ and tumor necrosis factor α. The Transwell chambers were incubated for 24 h to allow cell migration toward inflammatory cytokines. The cells were fixed with 10% formaldehyde, and cells on the upper side of the chamber, which had not migrated through the pore, were removed with cotton swabs. The remaining migrated cells were stained with DAPI. After washing, stained cells were counted under a fluorescence microscope .TMSC migration was analyzed using Transwell chambers with an 8-μm pore size. The cells 6, and 1 mM MgCl2 overnight at 37 °C. After washing, images were captured.Senescence-associated beta-galactosidase staining was conducted as previously described . BrieflyA colony formation assay was performed by plating the cells in 6-well plates (100 cells/well). After 2 weeks, the cells were washed twice, followed by fixation with ethanol 70%, 15 min), and staining with crystal violet (0.5%) at room temperature. After washing, images were captured.l-methylxanthine, 5 μg/mL insulin, and 60 μM indomethacin) for 3 weeks, and assessed by Oil Red O (Sigma-Aldrich) staining as an indicator of intracellular lipid accumulation. Before staining, the cells were fixed in 70% ethanol for 15 min at room temperature. The cells were then incubated in 2% Oil Red O reagent for 1 h at room temperature. To visualize lipid droplets, the excess stain was removed by washing with 70% ethanol and then distilled water. Osteogenic differentiation was induced by culturing the TMSCs for 3 weeks in osteogenic medium , with an examination of ECM calcification conducted by Alizarin Red S (Sigma-Aldrich) staining. Briefly, after fixation with 70% ethanol and washing with distilled water, the cells were incubated in 2% Alizarin Red solution for 15 min at room temperature and then washed numerous times with distilled water. Chondrogenic differentiation was induced using the micromass culture technique. Briefly, 10 μL of a concentrated MSC suspension (3 × 105 cells/mL) was plated into the center of each well and allowed to attach at 37 °C for 2 h. Chondrogenic medium , and 10 ng/mL transforming growth factor beta-1 (Sigma-Aldrich)) was gently overlaid so as to not detach the cell nodules, and the culture was maintained in the medium for 4 weeks before analysis. Chondrogenesis was confirmed by immunohistochemistry for collagen type-II staining. The sections were blocked with 10% horse serum, incubated with purified anti-mouse COL2A1 antibody (BD Biosciences) for 1 h, and then washed with PBS (pH 7.4). Antibody-bound cells were detected with a peroxidase substrate kit . The sections were then washed, counterstained with hematoxylin, and examined by light microscopy.The potential of TMSCs to differentiate into the adipogenic, osteogenic, and chondrogenic lineages was analyzed. Adipogenic differentiation was induced by culturing TMSCs in adipogenic medium , 100 μg/mL 3-isobutyl-Flow cytometric analysis was used to measure the total and active cell-surface ITGβ1 levels of the TMSCs. At least 50,000 cells  were incubated with phycoerythrin-conjugated monoclonal antibodies against human total and active ITGβ1 (BD Biosciences). The labeled cells were analyzed by flow cytometry using a FACSCaliber flow cytometer equipped with CellQuest Pro software (BD Biosciences).Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Tween 20 for 10 min, and blocked in 1% bovine serum albumin for 1 h at room temperature. The fixed cells were stained with purified rabbit anti-human ITGβ1 at 4 °C for 2 h and then incubated with phycoerythrin-conjugated goat anti-rabbit IgG  and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (e-Bioscience) for 1 h at room temperature. The cells were mounted with 4′,6-diamidino-2-phenylindole containing the VECTASHIELD (Vector Laboratories) mounting medium and examined using a confocal laser-scanning microscope.p value of <0.05 was considered statistically significant.The data are expressed as the mean ± standard deviation in all experiments. One-way analysis of variance , followed by Scheffé’s test, was conducted to detect significant differences between groups. A p < 0.05)  A. p < 0.05 on day 3; control 550 ± 40% vs. siTNS3 115 ± 3.3%, p < 0.05 on day 4). Moreover, the migration of TMSCs towards inflammatory cytokines was significantly suppressed by siTNS3 transfection  were examined. RT-qPCR revealed that the expression of TNS3 was significantly downregulated in siTNS3-transfected TMSCs B. Phase-sfection C–E.p16 and p21 are associated with cell cycle regulation and are frequently measured as markers of cellular senescence. After siTNS3 treatment, p16 and p21 (associated with cell cycle regulation) were increased at both the mRNA and protein levels. Among well-known pluripotency markers in stem cells, SOX2 expression was downregulated by TNS3 inhibition, whereas the expression of Nanog, c-Myc, and Oct-4 was not affected. Additionally, phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), protein kinase B , and c-Jun N-terminal kinase (JNK) was decreased after siTNS3 treatment A,B. CellGiven that critical factors involved in MSC proliferation are related to the senescence of MSCs ,13,14,15p < 0.05) (p < 0.05)  B. For os < 0.05) C. Treatm < 0.05) D. The ro < 0.05) E. RT-qPCed TMSCs F. These To investigate the effect of TNS3 on ITGβ1, integrin activity was measured after inhibition of TNS3. The proportions of cells expressing total and active ITGβ1 levels were detected in siTNS3-treated and siControl-treated TMSCs, respectively. Active ITGβ1-positive cells were decreased after siTNS3 treatment, whereas total form expression was not altered A. Consisp < 0.05 on day 3)  D. To furn day 3) E. Taken The proliferative capacity and multilineage differentiation potential of stem cells are critical for their clinical application. In a previous study , we showTensin is a cytoplasmic phosphoprotein located at focal adhesion sites. This multidomain protein can interact with several molecules and is divided into four types depending on the structure of its N-terminus . One of In this study investigating the role of TNS3 in TMSC functions, basal expression of TNS3 was found to be significantly more abundant in TMSCs than in ADSCs or BMSCs. Additionally, in siTNS3-transfected TMSCs, cell proliferation was significantly decreased, along with an increase in the G0/G1 phase and a decrease in the S phase, indicating cell cycle arrest. TNS3 plays an important role in the differentiation and proliferation of TMSCs, as adipogenic, osteogenic, and chondrogenic differentiation was reduced when TNS3 was downregulated in the cells. In particular, adipogenic differentiation was significantly reduced when TMSCs were treated with siTNS3.Integrins, transmembrane receptors composed of 18 alpha-subunits and 8 beta-subunits, are the central molecule in focal adhesion . IntegriIn the present study, ITGβ1 activity was significantly reduced after siTNS3 treatment. When ITGβ1 was blocked, its downstream signaling molecules  were decreased, which was similar to the results observed in TNS3 silencing. Moreover, the alterations in p16, p21, and SOX2 expression were consistent with the change observed in TNS3 inhibition. The findings indicate that TNS3 regulates the activity of ITGβ1 and influences the behavior of TMSCs.However, this study has several limitations. First, we examined whether TNS3 is significantly more expressed in TMSCs than in BMSCs or ADSCs, and thus TNS3 was the only tensin isoform investigated. The other tensin isoforms with structures similar to that of TNS3 may also have important functions in TMSCs. Particularly, because TNS4 is also known to play a complementary role to TNS3 ,35,36, fIn conclusion, our results show that TNS3 increased the proliferative capacity and differentiation potential of TMSCs by regulating ITGβ1 activity.