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We first sequenced and characterised the complete mitochondrial genome of Toxocara apodeme, then studied the evolutionary relationship of the species within Toxocaridae. The complete mitochondrial genome was amplified using PCR with 14 specific primers. The mitogenome length was 14303 bp in size, including 12 PCGs (encoding 3,423 amino acids), 22 tRNAs, 2 rRNAs, and 2 NCRs, with 68.38% A+T contents. The mt genomes of T. apodemi had relatively compact structures with 11 intergenic spacers and 5 overlaps. Comparative analyses of the nucleotide sequences of complete mt genomes showed that T. apodemi had higher identities with T. canis than other congeners. A sliding window analysis of 12 PCGs among 5 Toxocara species indicated that nad4 had the highest sequence divergence, and cox1 was the least variable gene. Relative synonymous codon usage showed that UUG, ACU, CCU, CGU, and UCU most frequently occurred in the complete genomes of T. apodemi. The Ka/Ks ratio showed that all Toxocara mt genes were subject to purification selection. The largest genetic distance between T. apodemi and the other 4 congeneric species was found in nad2, and the smallest was found in cox2. Phylogenetic analyses based on the concatenated amino acid sequences of 12 PCGs demonstrated that T. apodemi formed a distinct branch and was always a sister taxon to other congeneric species. The present study determined the complete mt genome sequences of T. apodemi, which provide novel genetic markers for further studies of the taxonomy, population genetics, and systematics of the Toxocaridae nematodes.
Population-wide restrictions during the COVID-19 pandemic may create barriers to mental health diagnosis. This study aims to examine changes in the number of incident cases and the incidence rates of mental health diagnoses during the COVID-19 pandemic.
Methods
By using electronic health records from France, Germany, Italy, South Korea and the UK and claims data from the US, this study conducted interrupted time-series analyses to compare the monthly incident cases and the incidence of depressive disorders, anxiety disorders, alcohol misuse or dependence, substance misuse or dependence, bipolar disorders, personality disorders and psychoses diagnoses before (January 2017 to February 2020) and after (April 2020 to the latest available date of each database [up to November 2021]) the introduction of COVID-related restrictions.
Results
A total of 629,712,954 individuals were enrolled across nine databases. Following the introduction of restrictions, an immediate decline was observed in the number of incident cases of all mental health diagnoses in the US (rate ratios (RRs) ranged from 0.005 to 0.677) and in the incidence of all conditions in France, Germany, Italy and the US (RRs ranged from 0.002 to 0.422). In the UK, significant reductions were only observed in common mental illnesses. The number of incident cases and the incidence began to return to or exceed pre-pandemic levels in most countries from mid-2020 through 2021.
Conclusions
Healthcare providers should be prepared to deliver service adaptations to mitigate burdens directly or indirectly caused by delays in the diagnosis and treatment of mental health conditions.
To accelerate high-intensity heavy-ion beams to high energy in the booster ring (BRing) at the High-Intensity Heavy-Ion Accelerator Facility (HIAF) project, we take the typical reference particle 238U35+, which can be accelerated from an injection energy of 17 MeV/u to the maximal extraction energy of 830 MeV/u, as an example to study the basic processes of longitudinal beam dynamics, including beam capture, acceleration, and bunch merging. The voltage amplitude, the synchronous phase, and the frequency program of the RF system during the operational cycle were given, and the beam properties such as bunch length, momentum spread, longitudinal beam emittance, and beam loss were derived, firstly. Then, the beam properties under different voltage amplitude and synchronous phase errors were also studied, and the results were compared with the cases without any errors. Next, the beam properties with the injection energy fluctuation were also studied. The tolerances of the RF errors and injection energy fluctuation were dictated based on the CISP simulations. Finally, the effect of space charge at the low injection energy with different beam intensities on longitudinal emittance and beam loss was evaluated.
This paper builds a patent-based knowledge graph, patent-KG, to represent the knowledge facts in patents for engineering design. The arising patent-KG approach proposes a new unsupervised mechanism to extract knowledge facts in a patent, by searching the attention graph in language models. The extracted entities are compared with other benchmarks in the criteria of recall rate. The result reaches the highest 0.8 recall rate in the standard list of mechanical engineering related technical terms, which means the highest coverage of engineering words.
To identify the clinical characteristics, treatment, and prognosis of relapsing polychondritis patients with airway involvement.
Methods
Twenty-eight patients with relapsing polychondritis, hospitalised in the First Hospital of Shanxi Medical University between April 2011 and April 2021, were retrospectively analysed.
Results
Fifty per cent of relapsing polychondritis patients with airway involvement had a lower risk of ear and ocular involvement. Relapsing polychondritis patients with airway involvement had a longer time-to-diagnosis (p < 0.001), a poorer outcome following glucocorticoid combined with immunosuppressant treatment (p = 0.004), and a higher recurrence rate than those without airway involvement (p = 0.004). The rates of positive findings on chest computed tomography and bronchoscopy in relapsing polychondritis patients with airway involvement were 88.9 per cent and 85.7 per cent, respectively. Laryngoscopy analysis showed that 66.7 per cent of relapsing polychondritis patients had varying degrees of mucosal lesions.
Conclusion
For relapsing polychondritis patients with airway involvement, drug treatment should be combined with local airway management.
The catechol-O-methyltransferase (COMT) gene is related to dopamine degradation and has been suggested to be involved in the pathogenesis of major depressive disorder (MDD). However, how this gene affects brain function properties in MDD is still unclear.
Methods:
Fifty patients with MDD and 35 cognitively normal participants underwent a resting-state functional magnetic resonance imaging scan. A voxelwise and data-drive global functional connectivity density (gFCD) analysis was used to investigate the main effects and the interactions of disease states and COMT rs4680 gene polymorphism on brain function.
Results:
We found significant group differences of the gFCD in bilateral fusiform area (FFA), post-central and pre-central cortex, left superior temporal gyrus (STG), rectal and superior temporal gyrus and right ventrolateral prefrontal cortex (vlPFC); abnormal gFCDs in left STG were positively correlated with severity of depression in MDD group. Significant disease × COMT interaction effects were found in the bilateral calcarine gyrus, right vlPFC, hippocampus and thalamus, and left SFG and FFA. Further post-hoc tests showed a nonlinear modulation effect of COMT on gFCD in the development of MDD. Interestingly, an inverted U-shaped modulation was found in the prefrontal cortex (control system) but U-shaped modulations were found in the hippocampus, thalamus and occipital cortex (processing system).
Conclusion:
Our study demonstrated nonlinear modulation of the interaction between COMT and depression on brain function. These findings expand our understanding of the COMT effect underlying the pathophysiology of MDD.
The small intestine is an important digestive organ and plays a vital role in the life of a pig. We tested the hypothesis that the length of the small intestine is related to growth performance and intestinal functions of piglets. A total of 60 piglets (Duroc × Landrace × Yorkshire), weaned at day 21, were fed an identical diet during a 28-day trial. At the end of the study, all piglets were sacrificed, dissected and grouped according to small intestine lengths (SILs), either short small intestine (SSI), middle small intestine (MSI) or long small intestine (LSI), respectively. Positive relationships between SIL and BW, average daily gain (ADG), average daily feed intake (ADFI) and gain-to-feed ratios (G : F) were observed. Final BW, ADG, ADFI and G : F significantly increased (P < 0.05) in MSI and LSI piglets compared with SSI piglets. Short small intestine and MSI had greater jejunal mucosa sucrase and alkaline phosphatase activities (P < 0.05) than LSI piglets. The mRNA level of solute carrier family 2 member 2 (Slc2a2) in the jejunal mucosa of SSI piglets was the greatest. The MSI piglets had a greater (P < 0.05) ileal villus height than other piglets and greater (P < 0.05) villus height-to-crypt depth ratios than LSI piglets. However, the LSI piglets had a greater (P < 0.05) ileal crypt depth than SSI piglets. No significant differences in duodenal, jejunal, caecal and colonic morphologies were detected among the groups. Moreover, luminal acetate, propionate, butyrate and total short-chain fatty acid contents were greater (P < 0.05) in SSI and MSI piglets than those in LSI piglets. In addition, there was greater serum glucose concentration in MSI piglets than other piglets. Serum albumin concentration in SSI piglets was the lowest. In conclusion, these results indicate that SIL was significantly positively associated with growth performance, and in terms of intestinal morphology and mucosal digestive enzyme activity, the piglets with a medium length of small intestine have better digestion and absorption properties.
Small intestinal epithelium homeostasis involves four principal cell types: enterocytes, goblet, enteroendocrine and Paneth cells. Epidermal growth factor (EGF) has been shown to affect enterocyte differentiation. This study determined the effect of dietary EGF on goblet, enteroendocrine and Paneth cell differentiation in piglet small intestine and potential mechanisms. Forty-two weaned piglets were used in a 2 × 3 factorial design; the major factors were time post-weaning (days 7 and 14) and dietary treatment (0, 200 or 400 µg/kg EGF supplementation). The numbers of goblet and enteroendocrine cells were generally greater with the increase in time post-weaning. Moreover, the supplementation of 200 µg/kg EGF increased (P < 0.01) the number of goblet and enteroendocrine cells in villus and crypt of the piglet small intestine as compared with the control. Dietary supplementation with 200 µg/kg EGF enhanced (P < 0.05) abundances of differentiation-related genes atonal homologue 1, mucin 2 and intestinal trefoil factor 3 messenger RNA (mRNA) as compared with the control. Piglets fed 200 or 400 µg/kg EGF diet had increased (P < 0.05) abundances of growth factor-independent 1, SAM pointed domain containing ETS transcription factor and pancreatic and duodenal homeobox 1 mRNA, but decreased the abundance (P < 0.01) of E74 like ETS transcription factor 3 mRNA as compared with the control. Animals receiving 400 µg/kg EGF diets had enhanced (P < 0.05) abundances of neurogenin3 and SRY-box containing gene 9 mRNA as compared with the control. The mRNA abundance and protein expression of lysozyme, a marker of Paneth cell, were also increased (P < 0.05) in those animals. As compared with the control, dietary supplementation with 200 µg/kg EGF increased the abundance of EGF receptor mRNA and the ratio of non-phospho(p)-β-catenin/β-catenin (P < 0.05) in villus epithelial cells at days 7 and 14. This ratio in crypt epithelial cells was higher (P < 0.05) on the both 200 and 400 µg/kg EGF groups during the same period. Our results demonstrated that dietary EGF stimulated goblet, enteroendocrine and Paneth cell differentiation in piglets during the post-weaning period, partly through EGFR and Wnt/β-catenin signalling.
The gut is composed of a single layer of intestinal epithelial cells and plays important roles in the digestion and absorption of nutrients, immune and barrier functions and amino acid metabolism. Weaning stress impairs piglet intestinal epithelium structural and functional integrities, which results in reduced feed intake, growth rates and increased morbidity and mortality. Several measures are needed to maintain swine gut development and growth performance after weaning stress. A large body of evidence indicates that, in weaning piglets, glutamine, a functional amino acid, may improve growth performance and intestinal morphology, reduce oxidative damage, stimulate enterocyte proliferation, modulate cell survival and death and enhance intestinal paracellular permeability. This review focuses on the effects of glutamine on intestinal health in piglets. The aim is to provide evidentiary support for using glutamine as a feed additive to alleviate weaning stress.
Feeding ruminants a high-grain (HG) diet is a widely used strategy to improve milk yield and cost efficiency. However, it may cause certain metabolic disorders. At present, information about the effects of HG diets on the systemic metabolic profile of goats and the correlation of such diets with rumen bacteria is limited. In the present study, goats were randomly divided into two groups: one was fed the hay diet (hay; n = 5), while the other was fed HG diets (HG; n = 5). On day 50, samples of rumen contents, peripheral blood serum and liver tissues were collected to determine the metabolic profiles in the rumen fluid, liver and serum and the microbial composition in rumen. The results revealed that HG diets reduced (P < 0.05) the community richness and diversity of rumen microbiota, with an increase in the Chao 1 and Shannon index and a decrease in the Simpson index. HG diets also altered the composition of rumen microbiota, with 30 genera affected (P < 0.05). Data on the metabolome showed that the metabolites in the rumen fluid, liver and serum were affected (variable importance projection > 1, P <0.05) by dietary treatment, with 47, 10 and 27 metabolites identified as differentially metabolites. Pathway analysis showed that the common metabolites in the shared key pathway (aminoacyl-transfer RNA biosynthesis) in the rumen fluid, liver and serum were glycine, lysine and valine. These findings suggested that HG diets changed the composition of the rumen microbiota and metabolites in the rumen fluid, liver and serum, mainly involved in amino acid metabolism. Our findings provide new insights into the understanding of diet-related systemic metabolism and the effects of HG diets on the overall health of goats.
Nitrogen (N) application and irrigation to winter wheat may decrease leaf temperature and enhance photosynthesis: as a result, more photosynthates will be allocated to the grains, resulting in higher grain yields. To investigate this hypothesis, a 2-year field study was conducted with three levels of N fertilizer application (no fertilizer, N0; 240 kg N/ha, N1; 360 kg N/ha, N2) and two different water regimes (rainfed with no irrigation, R; irrigation at the over-wintering, stem elongation and grain filling stages, W). The results show that both N application and supplemental irrigation significantly increased grain yield with increases in both grain number/m2 and the 1000-grain weight, viz., WN2>WN1>WN0>RN2>RN1>RN0. In addition, application of N under both water regimes significantly increased flag leaf area, above-ground biomass and single stem productivity and decreased leaf temperature, which led to an increase in net photosynthesis rates and ribulose bisphosphate (RuBP) carboxylase activity. Moreover, analysis of the chlorophyll α fluorescence transient showed that N fertilizer application and supplemental irrigation significantly increased electron donor and acceptor performance of the photosystem II reaction centre.
Iron-deficiency anemia is a public health concern that frequently occurs in pregnant mammals and neonatal offspring. Ferrous N-carbamylglycinate chelate (Fe-CGly) is a newly designed iron fortifier with proven effects in iron-deficient rats and weanling piglets. However, the effects of this new compound on pregnant mammals are unknown. Therefore, this experiment was conducted to evaluate the effects of Fe-CGly on sow reproductive performance and iron status of both sows and neonatal piglets. A total of 40 large-white sows after second parity were randomly assigned to two groups (n=20). They were receiving a diet including 80 mg Fe/kg as FeSO4 or Fe-CGly, respectively, from day 85 of gestation to parturition. The serum (day 110 of pregnancy) and placentas of sows were sampled. Litter size, mean weight of live born piglets, birth (live) litter weight, number of live born piglets, and the number of still-born piglets, mummies, and weak-born piglets were recorded. Once delivered, eight litters were randomly selected from the 20 litters per treatment, and one new-born male piglet (1.503±0.142 kg) from each selected litter was slaughtered within 3 h after birth from the selected litters, without colostrum ingestion. The serum, longissimus muscle, liver and kidneys of the piglets were collected. The iron status of the serum samples and the messenger RNA level of iron-related genes in the placenta, liver and kidney were analyzed. The results showed that litter weight of live born piglets was higher (P=0.030) in the Fe-CGly group (19.86 kg) than in the FeSO4 group (17.34 kg). Fe-CGly significantly increased placental iron concentration (P<0.05) of sows. It also significantly increased iron saturation and reduced the total iron-binding capacity of piglets (P<0.05) at birth. However, the results revealed that supplementation of Fe-CGly in sows reduced liver and kidney iron concentration of neonatal piglets (P<0.05), indicating decreased iron storage. In addition, the concentration of iron in the colostrum was not significantly changed. Therefore, the present results suggested that replacement of maternal FeSO4 supplement with Fe-CGly in the late-gestating period for sows could improve litter birth weight, probably via enhanced iron transportation in the placenta.
Fruit flies in the genus Bactrocera are global, economically important pests of agricultural food crops. However, basic life history information about these pests, which is vital for designing more effective control methods, is currently lacking. Artificial diets can be used as a suitable replacement for natural host plants for rearing fruit flies under laboratory conditions, and this study reports on the two-sex life-table parameters of four Bactrocera species (Bactrocera correcta, Bactrocera dorsalis, Bactrocera cucurbitae, and Bactrocera tau) reared on a semi-artificial diet comprising corn flour, banana, sodium benzoate, yeast, sucrose, winding paper, hydrochloric acid and water. The results indicated that the larval development period of B. correcta (6.81 ± 0.65 days) was significantly longer than those of the other species. The fecundity of B. dorsalis (593.60 eggs female−1) was highest among the four species. There were no differences in intrinsic rate of increase (r) and finite rate of increase (λ) among the four species. The gross reproductive rate (GRR) and net reproductive rate (R0) of B. dorsalis were higher than those of the other species, and the mean generation time (T) of B. cucurbitae (42.08 ± 1.21 h) was longer than that of the other species. We conclude that the semi-artificial diet was most suitable for rearing B. dorsalis, due to its shorter development time and higher fecundity. These results will be useful for future studies of fruit fly management.
Retreatment of tuberculosis (TB) often fails in China, yet the risk factors associated with the failure remain unclear. To identify risk factors for the treatment failure of retreated pulmonary tuberculosis (PTB) patients, we analyzed the data of 395 retreated PTB patients who received retreatment between July 2009 and July 2011 in China. PTB patients were categorized into ‘success’ and ‘failure’ groups by their treatment outcome. Univariable and multivariable logistic regression were used to evaluate the association between treatment outcome and socio-demographic as well as clinical factors. We also created an optimized risk score model to evaluate the predictive values of these risk factors on treatment failure. Of 395 patients, 99 (25·1%) were diagnosed as retreatment failure. Our results showed that risk factors associated with treatment failure included drug resistance, low education level, low body mass index (<18·5), long duration of previous treatment (>6 months), standard treatment regimen, retreatment type, positive culture result after 2 months of treatment, and the place where the first medicine was taken. An Optimized Framingham risk model was then used to calculate the risk scores of these factors. Place where first medicine was taken (temporary living places) received a score of 6, which was highest among all the factors. The predicted probability of treatment failure increases as risk score increases. Ten out of 359 patients had a risk score >9, which corresponded to an estimated probability of treatment failure >70%. In conclusion, we have identified multiple clinical and socio-demographic factors that are associated with treatment failure of retreated PTB patients. We also created an optimized risk score model that was effective in predicting the retreatment failure. These results provide novel insights for the prognosis and improvement of treatment for retreated PTB patients.
Schizophrenia patients have a higher prevalence of type 2 diabetes mellitus with impaired glucose tolerance (IGT) than normals. We examined the relationship between IGT and clinical phenotypes or cognitive deficits in first-episode, drug-naïve (FEDN) Han Chinese patients with schizophrenia.
Method
A total of 175 in-patients were compared with 31 healthy controls on anthropometric measures and fasting plasma levels of glucose, insulin and lipids. They were also compared using a 75 g oral glucose tolerance test and the homeostasis model assessment of insulin resistance (HOMA-IR). Neurocognitive functioning was assessed using the MATRICS Consensus Cognitive Battery (MCCB). Patient psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS).
Results
Of the patients, 24.5% had IGT compared with none of the controls, and they also had significantly higher levels of fasting blood glucose and 2-h glucose after an oral glucose load, and were more insulin resistant. Compared with those patients with normal glucose tolerance, the IGT patients were older, had a later age of onset, higher waist or hip circumference and body mass index, higher levels of low-density lipoprotein and triglycerides and higher insulin resistance. Furthermore, IGT patients had higher PANSS total and negative symptom subscale scores, but no greater cognitive impairment except on the emotional intelligence index of the MCCB.
Conclusions
IGT occurs with greater frequency in FEDN schizophrenia, and shows association with demographic and anthropometric parameters, as well as with clinical symptoms but minimally with cognitive impairment during the early course of the disorder.
Ectropis grisescens Warren and Ectropis obliqua (Prout) are two morphologically similar sibling species with overlapping ranges. In this study, manipulative laboratory experiments were conducted to examine the possibility of reproductive interference in sympatric populations of E. grisescens and E. obliqua and the potential consequences of the mating interaction. Our results showed that the presence of males or females of different species could incur mating interference and significant reduction of F1 offspring. The reduction was not significant relevant to the initial relative abundance of E. grisescens and E. obliqua. Detailed observations of mating opportunity showed that female mating frequencies of both species were not significantly affected by the absolute species density, but the mating success of E. obliqua females with conspecific males depended on species ratio. In addition, adding males to the other species resulted in lower number of offspring suggesting that the males’ behaviour might be linked with mating interference. Males of both E. grisescens and E. obliqua could interfere the intraspecific mating of the other species, but the impact of the mating interference differed. These combined data indicated that asymmetric reproductive interference existed in E. grisescens and E. obliqua under laboratory conditions, and the offspring of the mixed species were significantly reduced. The long term outcome of this effect is yet to be determined since additional reproductive factors such as oviposition rate and progeny survival to adulthood may reduce the probability of demographic displacement of one species by the other in overlapping niches.
Most previous studies on the effects of lipopolysaccharide (LPS) in pigs focused on the body’s immune response, and few reports paid attention to body metabolism changes. To better understand the glucose metabolism changes in skeletal muscle following LPS challenge and to clarify the possible mechanism, 12 growing pigs were employed. Animals were treated with either 2 ml of saline or 15 µg/kg BW LPS, and samples were collected 6 h later. The glycolysis status and mitochondrial function in the longissimus dorsi (LD) muscle of pigs were analyzed. The results showed that serum lactate content and NADH content in LD muscle significantly increased compared with the control group. Most glycolysis-related genes expression, as well as hexokinase, pyruvate kinase and lactic dehydrogenase activity, in LD muscle was significantly higher compared with the control group. Mitochondrial complexes I and IV significantly increased, while mitochondrial ATP concentration markedly decreased. Significantly increased calcium content in the mitochondria was observed, and endoplasm reticulum (ER) stress has been demonstrated in the present study. The results showed that LPS treatment markedly changes glucose metabolism and mitochondrial function in the LD muscle of pigs, and increased calcium content induced by ER stress was possibly involved. The results provide new clues for clarifying metabolic diseases in muscle induced by LPS.
In this study, epidemiological factors of sparganosis cases reported in mainland China from 1959 to December 2012 were analysed. A total of 1061 valid cases were distributed throughout most of the provinces of mainland China, with most cases occurring in Southern and Eastern China. The average age of patients was 29 years (range 0–80 years). Modes of transmission to humans were via contact (54·6%), mainly by application of frog meat as a poultice, foodborne (33·8%), mainly through ingesting frogs or snakes, and waterborne (11·5%) through drinking raw water. The tissue/organs involved were subcutaneous/muscle (43·1%), eyes (31·0%), central nervous system (CNS) (17·9%), urogenital system (3·9%) and visceral organs (3·2%). Obvious differences existed in main risk factors for different areas. Close correlation was found between tissue/organs and risk factors. Main modes of transmission changed during the past decades, from contact (83·8% pre-1979) to foodborne (63·9% post-2000). The tissue/organs involved also changed at the same time. Cases involving eyes fell from 50·0% pre-1979 to 8·3% post-2000, and cases involving CNS increased from 0% pre-1979 to 47·8% post-2000. These results illustrate that China is one of the main epidemic countries of sparganosis in the world. Consumption of frog/snake meat was the main risk factor, although application of frog flesh as a poultice was the main risk factor before 2000. Sparganosis has become one of the neglected but important foodborne/waterborne parasitic diseases in mainland China.
Convergent studies have highlighted the dysfunction of the amygdala, prefrontal cortex and hippocampus in post-traumatic stress disorder (PTSD). However, only a few studies have investigated the functional connectivity between brain regions in PTSD patients during the resting state, which may improve our understanding of the neuropathophysiology of PTSD. The aim of this study was to investigate patterns of whole-brain functional connectivity in treatment-naive PTSD patients without co-morbid conditions who experienced the 8.0-magnitude earthquake in the Sichuan province of China.
Method
A total of 72 PTSD patients and 86 trauma-exposed non-PTSD controls participated in the resting-state functional magnetic resonance imaging study. All these subjects were recruited from the disaster zone of the 2008 Sichuan earthquake. Functional connectivities between 90 paired brain regions in PTSD patients were compared with those in trauma-exposed non-PTSD controls. Furthermore, Pearson correlation analysis was performed between significantly abnormal connectivities in PTSD patients and their clinician-administered PTSD scale (CAPS) scores.
Results
Compared with non-PTSD controls, PTSD patients showed weaker positive connectivities between the middle prefrontal cortex (mPFC) and the amygdala, hippocampus, parahippocampal gyrus and rectus, as well as between the inferior orbitofrontal cortex and the hippocampus. In addition, PTSD patients showed stronger negative connectivity between the posterior cingulate cortex (PCC) and the insula. The CAPS scores in PTSD patients correlated negatively with the connectivity between the amygdala and the mPFC.
Conclusions
PTSD patients showed abnormalities in whole-brain functional connectivity, primarily affecting the connectivities between the mPFC and limbic system, and connectivity between the PCC and insula.