Pattern and extent of intrahepatic infiltration of perihilar cholangiocarcinoma - A case-control study based on liver panoramic digital pathology.

BACKGROUND: The extent of intrahepatic infiltration of perihilar cholangiocarcinoma (PHCC) remains unclear. This research aimed to explore the pattern and extent of intrahepatic infiltration of PHCC to guide surgical treatment and pathological research. MATERIALS AND METHODS: This study included 62 patients diagnosed with PHCC who underwent major hepatectomy. A whole-mount digital liver pathology system (WDLPS) for hepatectomy specimens greater than 10 × 10 cm was used to panoramically assess the intrahepatic infiltration extent of PHCC. RESULTS: The distal intrahepatic infiltration (DIHI) and radial liver invasion (RLI) were important parts of intrahepatic infiltration for PHCC explored by WDLPS. The study confirmed that 75.8% of PHCCs had RLI and the infiltration distance in all patients were within 15,000 µm, 62.9% of PHCCs had DIHI greater than 1 cm away from the main tumor in liver parenchyma. The recurrence-free survival rates and overall survival rates of patients with DIHI were poorer than the patients without DIHI (P＜0.0001, P=0.0038). Arterial invasion on the resected side could be an excellent predictor. A total of 105 liver lobes were resected from 62 PHCC patients. The invasion rates of the left lateral, left medial, right anterior, and right posterior lobe of PHCC were 79%, 100, 100%, and 69% respectively. CONCLUSION: The presence of DIHI in most PHCCs was a significant predictor of poor postoperative recurrence and survival. Based on the extent of intrahepatic infiltration, minor hepatectomy was not suitable as the curative surgery for PHCC. Major hepatectomy and liver transplantation were the ideal radical treatment.

First Report of Didymella pomorum Causing Leaf Blight on Angelica sinensis in China.

Angelica sinensis (Oliv.) Diels, is a perennial herbaceous plant of the Umbelliferae family. It has a long history of cultivation and is highly valued as a traditional Chinese medicine in China (Zhang et al. 2012). In September 2023, leaf blight on A. sinensis with an average disease incidence of 56% was recorded in an approximately 6.7-ha production field in Lijiang, Yunnan province, China (26.8215°N, 100.2369°E). At first, small, chlorotic lesions appeared on the leaves. They subsequently increased in density and gradually merged, causing leaves to yellow and wither. Ultimately the blight casused death of the entire foliage. In order to identify the causal agent, cross-sectional segments (5×5 mm2) were cut from the edge of leaf lesions, surface disinfected with a 1% sodium hypochlorite solution for 3 min and rinsed three times with sterile distilled water. They were subsequently placed on potato dextrose agar (PDA) plates and incubated for 3 days under a 12-h photoperiod at 28℃. A total of ten isolates with similar morphological characteristics were obtained by single spore isolation. After 10 days of incubation on PDA, the colony morphology of these isolates was characterized by a brownish central area with a white edge. Aged colonies became wrinkled in the center of the colony. Conidia (n = 30) were elliptical and brown, with a size range of 4.11 to 6.55 µm (average 5.37±0.74 µm) × 3.17 to 4.62 µm (average 3.92±0.43 µm). Chlamydospores (n = 30) formed chains in series, spherical or elliptical in shape, ranging from yellow-brown to dark brown, with a size range of 12.30 to 13.70 µm (average 12.98±0.46 µm) × 4.20 to 5.30 µm (average 4.63±0.26 µm). The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of RNA polymerase II (RPB2), and the 28S nuclear ribosomal large subunit rRNA (LSU) region of two isolates were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), fRPB2-5F/fRPB2-7cR (Liu et al. 1999), and LR0R/LR5 (Schoch et al. 2012), respectively. These amplicons were sequenced bidirectionally and assembled. The two isolates produced the same nucleotide sequences, and the sequences of a representative isolate (AsDp1) were deposited in GenBank. BLASTn analyses showed that the ITS (PP510616), RPB2 (PP526010), and LSU (PP550143) sequences of isolate AsDp1 were 100%, 99.66%, and 100% identical with those of Didymella pomorum ex-type isolate CBS 354.52 (MH857081, KT389616, and MH868616), respectively. A phylogenetic tree was constructed based on the ITS, RPB2, and LSU concatenated nucleotide sequences using the maximum likelihood method in MEGAX. Isolate AsDp1 was clustered with four D. pomorum isolates. According to the morphological and nucleotide sequences analyses, isolate AsDp1 was identified as D. pomorum (Chen et al. 2015). To determine pathogenicity, 1-year-old A. sinensis plants (approximately 20 cm tall) grown in 7-liter pots filled with sterilized field soil were sprayed until runoff with a 1×106 conidia/ml suspension of isolate AsDp1 onto the foliage, while control plants were sprayed with sterile water. All plants were cultivated under a 12-h photoperiod at 25℃. The pathogenicity tests were performed in triplicate with ten plants in each treatment. After fifteen days, numerous chlorotic lesions appeared on the leaves of all inoculated plants. The symptoms were similar to those found on naturally infected plants in the field, while the control plants remained asymptomatic. Subsequently, D. pomorum was reisolated from the diseased leaves, and the identity was confirmed based on its ITS sequence and morphological characteristics. D. pomorum causing stem canker on Rosa spp. was reported in Canada (Ilyukhin 2022). To our knowledge, this is the first report of D. pomorum causing leaf blight on A. sinensis in China. This etiological finding will potentially pave the way for the development of control strategies of this disease.

First report of Tomato Zonate Spot Virus infecting Belamcanda chinensis in China.

Tomato zonate spot virus (TZSV, Orthotospovirus tomatozonae, genus Orthotospovirus, family Tospoviridae) was first reported to infect tomato (Solanum lycopersicum) in China in 2008 (Dong et al. 2008). Belamcanda chinensis (L.) Redouté is a perennial herbaceous medicinal plant of the family Iridaceae, which is widely distributed in China. Its rhizome contains abundant active components, mainly including flavonoids, and has antibacterial, anticancer, and antioxidative effects. In July 2023, four B. chinensis plants with virus-like symptoms were collected in Fuyuan County, Yunnan Province in China. The diseased leaves showed chlorosis and ringspots (Fig. S1). Spherical virus particles with a diameter of 80-100 nm were observed in the saps of diseased leaves under a transmission electron microscope (Fig. S2). The presence of an orthotospovirus was confirmed by the previously reported method to amplify the partial sequence (312 nt) of L segment (Huang et al. 2018) (Fig. S3). BLASTn analysis showed that the obtained 312-nt sequence was 95.62% nucleotide identity with TZSV tomato-YN isolate (accession no. NC_010491.1). To obtain the complete genome of this isolate, total RNA from symptomatic leaves of two single diseased B. chinensis were extracted using Hipure Universal RNA Mini Kit (Magen Biotech) and subjected to high-throughput sequencing with a NovaPE150 (Illumina, USA) at MAGIGENE (Shenzhen, China). A total of 41,144,571 clean reads were obtained after removing low quality reads. Quality-controlled, qualified reads were assembled into contigs using Megahit v1.1.2 software. Thirteen contigs shared nucleotide identity ranging 86.94%-97.73% with the L, S, and M segments of TZSV using BLASTn searches online (https://blast.ncbi.nlm.nih.gov/Blast.cgi). In addition, no contigs were mapped to other viral (taxid:10239) and viroidal (taxid:12884) sequences in GenBank Databases. The full-length L, M, and S RNA segments of TZSV-Bc isolate was determined tbe 8917 nt (PP314222), 4718 nt (PP314223) and 3213 nt (PP314224), respectively. These segments were validated by RT-PCR, and Sanger sequencing. They shared nucleotide sequence identities of 95.9%, 97.2%, and 93.1% of the L (NC_010491.1), M (NC_010490.1), and S (NC_010489.1) segments, of the TZSV tomato-YN isolate, respectively. Compared to the TZSV tomato-YN isolate, there exists a missing segment with 113 nt in the intergenic region of S RNA and a segment with 199 nt in M RNA. To further confirm the TZSV infection on B. chinensis, three primers pairs Tosp10/ Tosp11， Tosp5/Tosp6, and NSs-F/NSs-R were tested by RT-PCR for TZSV based on the previous report (Dong et al, 2008). The sequences of amplicons shared >99% nucleotide identity with the corresponding TZSV-Bc isolate sequences. Total of 14 B. chinensis samples were detected with the primer pair N-F/N-R (5'-ATGTCTAACGTCCGGAGTTTAACA-3'/ 5'-AAAAGACAGATCATTGCTGCTCTT-3') by One Step RT-PCR, 6 samples (42.85%) showed the positive results. The mechanical inoculation and RT-PCR detection confirmed TZSV-Bc isolate can infect N. bethamiana. So far, tomato zonate spot virus has been detected in different plants including tobacco (N. tabacum) (Huang et al. 2017), sticktight (Bidens pilosa) (Xu et al. 2022), pepper (Capsicum annuum) (Li et al. 2023) in China. To our knowledge, it is the first report of TZSV naturally infecting B. chinensis plants, which enriches information on the host range of TZSV and will be helpful for disease management.

First report of Alternaria alternata causing leaf spot on Polygonatum kingianum in China.

Polygonatum kingianum is a Chinese herbal medicine that belongs to the genus Polygonatum of the family Liliaceae. In June 2023, Polygonatum kingianum Coll. et Hemsl. in nurseries in Qujing, Yunnan Province, China, showed irregular brown spots on the leaves, whole leaf necrosis, and plant death in serious cases, with an incidence of 10-20% (Fig. S1). To identify the pathogens of P. kingianum, six diseased samples were collected from nurseries with 0.6 acre. These diseased sample leaves were soaked in 0.1% HgCl2 for 1 min and 75% ethanol for 2 min and then rinsed thrice with sterile water. Treated leaves were cut into small pieces (5×5 mm) and cultured on potato dextrose agar (PDA) for five days at 28°C. Total thirteen fungal strains were isolated from PDA medium. The nuclear ribosomal internal transcribed spacer of ribosomal DNA (ITS rDNA) region of these 13 strains was amplified by polymerase chain reaction (PCR) using universal primers ITSI/ITS4 (White et al. 1990). Sequencing and BLAST of the ITS region on NCBI showed that 11 out of 13 fungal strains belonged to the genus Alternaria, with an identity ≥99%. We selected one of the Alternaria strains, HJ-A1, for further study. The HJ-A1 colony appeared grayish brown white-to-gray with a flocculent texture on the front side and a dark gray underside on the PDA medium (Fig. S1). The conidiophores appeared brown, either single or branched, and produced numerous short conidial chains. The conidia were obclavate to obpyriform or ellipsoid in shape and contained 1-4 transverse septa and 0-2 oblique septa. The conidial diameter was 27.30µm in length and 12.27µm in width. (Fig. S1). To further determine the species of HJA1, the genomic DNA of HJ-A1 was extracted using the Lysis Buffer for PCR (AG, Hunan, China). Four Alternaria genomic DNA regions including the ITS, translation elongation factor 1-α gene (TEF1-α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and Alternaria major allergen gene (Alt a1) were amplified by PCR using the primers as previously reported (Woudenberg et al. 2013, Hong et al. 2005). Sequence analysis revealed that the ITS (484bp) of HJ-A1 (NCBI No. PP082633), TEF1-α (267bp) of HJ-A1 (NCBI No. PP419893), GAPDH (582bp) of HJ-A1 (NCBI No. PP419892), and Alt a1 (522bp) of HJ-A1 (NCBI No. PP228046) shared the highest identity with A. alternata respectively (99≥%). A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, GAPDH, TEF, and Alt a1 using MEGA 7. The results showed that HJ-A1 strain clustered with A. alternate (Fig. S2). The pathogenicity of HJ-A1 was tested according to Koch's postulates by inoculating HJ-A1 conidia suspension (2×105 conidia/mL) into leaves of 1-year-old P. kingianum, with sterile water as a control. Each treatment group included 3 plants with 3 replicates. The tested plants were planted in a phytotron at 28℃ and 90% humidity. Three days after inoculation, symptoms similar to those under natural conditions were observed in the HJ-A1-inoculated plants, whereas no symptoms were observed in the control plants (Fig. S1). The same fungal strains were re-isolated from inoculated leaves and identified by morphologically and sequence of ITS. Previous studies showed that Alternaria alternata funji cause many plant diseases, such as fig fruit rot (Latinovic N et al. 2014)，daylily leaf spot (Huang D et al. 2022), fruit blight on sesame (Cheng H et al. 2021)，leaf spot of Cynanchum atratum Bunge (Sun H et al. 2021) and so on. To our knowledge, this is the first report of A. alternata causing P. kingianum leaf spot in China. The discovery of this pathogen will help to guide the protection and control of P. kingianum disease.

Safety and benefit of modified mesohepatectomy in the treatment of bilobar involvement congenital biliary dilatation.

BACKGROUND: The diseased bile duct in bilobar congenital biliary dilatation is extensive and often requires major hepatectomy or liver transplantation associated with a higher risk. We aimed to evaluate the safety and benefit of modified mesohepatectomy, in comparison with trisectionectomy, to treat bilobar congenital biliary dilatation. METHODS: This study included 28 patients with type IV and V bilobar congenital biliary dilatation. An innovative mesohepatectomy comprising the hepatectomy technique beyond the P/U point and bile duct shaping was applied to 14 patients to address the extensively diseased bile duct and difficulty in hepaticojejunostomy. Another 14 patients received trisectionectomy. The perioperative and long-term outcomes of these patients were compared. RESULTS: The ratio of residual liver volume to standard liver volume in the mesohepatectomy group was higher (78.68% vs. 40.90%, p = 0.005), while the resection rate of the liver parenchyma was lower (28.25% vs. 63.97%, p = 0.000), than that in trisectionectomy group. The mesohepatectomy group had a lower severe complication (>Clavein III, 0% vs. 57.70%, p = 0.019) and incidence of posthepatectomy liver failure (7.14% vs. 42.86%, p = 0.038). No significant difference was observed in blood loss and bile leakage (p > 0.05). All the patients in the mesohepatectomy group achieved optimal results in the long-term follow-up. CONCLUSIONS: mesohepatectomy provides an efficient treatment option for bilobar congenital biliary dilatation and can achieve radical resection, retain more liver parenchyma, and reduce the difficulty of hepaticojejunostomy, especially for patients that are not eligible for major hepatectomy and liver transplantation.

Predicting the outcomes of hepatocellular carcinoma downstaging with the use of clinical and radiomics features.

BACKGROUND: Downstaging of hepatocellular carcinoma (HCC) makes it possible for patients beyond the criteria to have the chance of liver transplantation (LT) and improved outcomes. Thus, a procedure to predict the prognosis of the treatment is an urgent requisite. The present study aimed to construct a comprehensive framework with clinical information and radiomics features to accurately predict the prognosis of downstaging treatment. METHODS: Specifically, three-dimensional (3D) tumor segmentation from contrast-enhanced computed tomography (CT) is employed to extract spatial information of the lesions. Then, the radiomics features within the segmented region are calculated. Combining radiomics features and clinical data prompts the development of feature selection to enhance the robustness and generalizability of the model. Finally, we adopt the support vector machine (SVM) algorithm to establish a classification model for predicting HCC downstaging outcomes. RESULTS: Herein, a comparative study was conducted on three different models: a radiomics features-based model (R model), a clinical features-based model (C model), and a joint radiomics clinical features-based model (R-C model). The average accuracy of the three models was 0.712, 0.792, and 0.844, and the average area under the receiver-operating characteristic (AUROC) of the three models was 0.775, 0.804, and 0.877, respectively. CONCLUSIONS: The novel and practical R-C model accurately predicted the downstaging outcomes, which could be utilized to guide the HCC downstaging toward LT treatment.

First Report of Root Rot on Paris polyphylla var. yunnanensis caused by Fusarium solani in Yunnan, China.

Paris polyphylla var. yunnanensis is a perennial herb with diverse chemical components having wide-ranging pharmacological effects. The demand for P. polyphylla var. yunnanensis as a raw material increases greatly and currently exceeds 1,000 tons per year (Zhou et al. 2021). In September 2021, root rot was observed on P. polyphylla var. yunnanensis in Mangshi, Yunnan province, China. Average disease incidences in the fields reached 15%, with diseased plants exhibiting yellowing and wilting leaves, as well as browning and rotting roots. Cross sections (5 × 5 mm2) cut from the margin of symptomatic and asymptomatic root tissues were surface-sterilized for 30 s with 75% ethanol, followed by 180 s with 1% sodium hypochlorite. After rinsing thrice with sterile distilled water, the fragments were transferred to potato dextrose agar (PDA) plates and incubated at 28°C in the dark. Ten isolates were obtained, and single spore isolation was performed. These isolates showed similar morphological characters, with colonies ranging in color from white to pale cream and sparse mycelia. Conidia were produced on the top or side of phialides. Microconidia were oval or reniform, 0- or 1-septate, with a diameter of 5.1-10.7 µm × 1.6-3.9 µm (average 7.6 µm × 2.8 µm) (n=30). The macroconidia were straight to slightly curved or sickle-shaped, 3- to 5-septate, with a diameter of 15.1-27.9 µm × 2.8-4.0 µm (average 21.0 µm × 3.6 µm). Chlamydospores were smooth, nearly round, and 3.3-6.6 (average 4.9) µm in diameter. Genomic DNA were extracted from mycelia of the two isolates. The nuclear ribosomal internal transcribed spacer (ITS), translation elongation factor 1 alpha (EF1α), and the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2) were amplified with the primer pairs of ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. These two isolates exhibited the same nucleotide sequences (ITS, OP646781; EF1α, OP661172; RPB2, OP661173), with BLASTn analyses showing 100%, 99.66%, and 99.65% identity, respectively, with Fusarium solani (syn. Neocosmospora solani) (Crespo et al. 2019) strain NRRL 43474 (ITS, EF453097; EF1α, EF452945; RPB2, EF469984). A phylogenetic tree was constructed using MEGAX based on the nucleotide sequences of ITS, EF1α, and RPB2, using the maximum likelihood method. The isolate was classified into the F. solani clade. According to the morphology and sequence analyses, the isolate was identified as F. solani (Chehri et al. 2015), and named PpFs1. To test the pathogenicity of the isolate PpFs1, the roots of four years old P. polyphylla var. yunnanensis plants were dipped in 107 spore/mL suspension filtered from potato dextrose broth (PDB) for 30 min, while control roots were dipped in sterile water. After inoculation, all plants were transplanted in pots filled with sterile soil and kept at 25°C with a 12/12-h light/darkness photoperiod. Six plants were used for each treatment, and repeated thrice. Two months after inoculation, the infected plants showed wilted leaves and rotted roots, while controls remained asymptomatic. PpFs1, identified by morphology and ITS, was re-isolated from infected plants, and was found to comply with Koch's postulates. To the best of our knowledge, F. oxysporum and F. concentricum causes Paris polyphylla var. Chinensis stem rot in China. But this is the first report of root rot on P. polyphylla var. yunnanensis being caused by F. solani in Yunnan, China.

First Report of Penicillium subrubescens Causing Root Rot of Knoxia roxburghii in China.

Knoxia roxburghii (syn. Knoxia valerianoides), locally known as 'Zi Daji', is a perennial herb that belongs to the Rubiaceae family, cultivated in different areas of China and recognized for its medicinal properties in traditional Chinese medicine (Chen et al. 2022). In 2021, root rot was observed during summer on K. roxburghii in Xiangyun and Dali, Yunnan Province (25°25'N, 100°40'E), China. Root rots were characterized by dark brown tissue from stem base to root, loss of vitality in tender leaves and wither. Three symptomatic root samples of K. roxburghii collected from different fields were rinsed with running water, and 0.5-1 cm2 fragments of diseased tissues were cut and surface-disinfested with 75% ethanol for 30 s, and 1% NaClO for 180 s. The fragments were then washed with sterile water, transferred to potato dextrose agar (PDA, 4.6%) and incubated at 28℃ in the dark for 3 days. A total of 13 isolates with consistent appearances were obtained by single spore isolation. These colonies on PDA showed gray and light brown obverse, and light green reverse after 10 days. The average growth rate was 3 mm per day. Conidia were nearly spherical or broadly elliptic, greyish-green, and 1.4-2.4×1.3-2.2 µm in size (n=50). The conidiophore has symmetrical or asymmetrical broom branches from the tip, with 2-4 small stems. The conidiophore branching patterns were predominantly biverticillate; stipes coarsely roughened, 80-210×2.6-3.0 µm; metulae were usually appressed verticils of 3-6, 6.4-12.5×1.6-3.0 µm; phialides were short and wide neck, 3.8-4.8×1.1-1.8 µm (n=30). The morphological characteristics of the fungus were identical to Penicillium (Mansouri et al. 2013). To further identify the isolate, one isolate (ByF10) was randomly selected for identification. DNA was extracted from mycelia using a simplified CTAB method. Primer pairs, ITS1/ITS4 and Bt2a/Bt2b were used to amplify the partial regions of rDNA internal transcribed spacer (ITS) and ß-tubulin (TUB), respectively (White et al. 1990; Glass and Donaldson 1999). Blast searches showed that the sequences of ITS (OQ954757) and TUB (OQ970059) of isolate ByF10 were 99% (MH865456) and 100% (KC797611) identical with P. subrubescens CBS 130205 and CBS 129617, respectively. A concatenated phylogenetic tree (ITS+TUB) constructed using the maximum likelihood method showed that ByF10 was closely grouped next to isolates of P. subrubescens. Pathogenicity test was carried out using 1-year-old healthy seedlings of K. roxburghii cv. Yunji-1 growing on autoclaved soil (n=10). Ten plants were inoculated with mycelial blocks (5 mm2), which were taken from the colony margins of a 10-day-old culture (PDA) colony, and placed on the roots near the soil. Five control plants were inoculated with non-colonized PDA plugs. The pathogenicity test was repeated three times. All plants were kept at 25℃, 70% relative air humidity, and 12 h light/12 h regime dark for 35 days. After that period 95% of inoculated plants showed typical symptoms of root browning. P. subrubescens was only re-isolated from the inoculated plants, and identified based on morphological and molecular characteristics. No symptoms were observed in the controls. To the best of our knowledge, this is the first report of P. subrubescens causing root rot on K. roxburghii in China and the world.

First report of Didymella curtisii causing leaf scorch on spider lily (Hymenocallis littoralis) in China.

Hymenocallis littoralis (Jacq.) Salisb, commonly known as spider lily, is a Amaryllidaceae species widely cultivated in southern China for ornamental and medicinal purposes (Anusha et al., 2016). In 2020, there was a devastating outbreak of leaf scorch of H. littoralis from July to September in Kunming city, Yunnan province of China (E102.8268°, N24.8371°), with 97% disease incidence. The initial spots were small and reddish-brown but gradually expanded to large irregular lesions with yellow centers. The leaves then turned yellow and withered from tip towards the petiole in the severely infected plants. For identification, leaf sections (5 × 5 mm2) cut from the margins of the lesions were surface-sterilized for 3 min with 1% sodium hypochlorite, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated at 28°C at a 12-h photoperiod for three days. One fungus was isolated from 90% of the samples, of which three monosporic isolates were selected using a method of agar dilution lineation separation. After 4 days of incubation, colonies were white-yellowish, and changed to beige-mustard after 7 days. The pycnidia were produced after 10 days incubation on oatmeal agar (OA), and they were black-brown, subglobose, and ostiolate. Chlamydospores mainly formed in the aerial mycelia, globose, often in chains, brown or pale. The conidia were ellipsoidal or clavate, (0-)1-3-septate, 2.1 to 10.8 × 1.0 to 3.2 µm (n=60). The total genomic DNA of three isolates was extracted from mycelia. The nuclear ribosomal internal transcribed spacer region (ITS), the second largest subunit of nuclear DNA-dependent RNA polymerase II (rpb2), the 28S nuclear ribosomal large subunit rRNA gene (LSU), and beta-tubulin gene (tub2) were amplified using the ITS1/ITS4 (White et al., 1990), fRPB2-5F/fRPB2-7cR (Liu et al., 1999), LR0R/LR5 (Schoch et al., 2012), and Btub2Fd/Btub4Rd (Woudenberg et al., 2009) primer pairs, respectively. The amplicons were cloned in the pMD19-T vector (Code No. 6013, Takara, Kusatsu, Japan) and sequenced bi-directionally. These three isolates had the same nucleotide sequences, one of which was submitted to NCBI (ITS, OM279485; rpb2, OM304305; LSU, OP800249; tub2, OQ108870). BLASTn analyses showed that ITS, rpb2, LSU, and tub2 were genetically 100%, 98.49%, 99.89%, and 97.89%, respectively, identical with MN973518, MT018130, MN943724, and MT005618 genes of Didymella curtisii strain CBS 288.29. Phylogenetic tree was constructed with MEGAX based on the nucleotide sequences of ITS, rpb2, LSU, and tub2 using the maximum likelihood method. The fungus isolated from diseased leaves of H. littoralis was grouped into the same clade with D. curtisii. According to the morphology and sequence analyses, the isolate was D. curtisii (Chen et al., 2015) and was named isolate HlDc1. To confirm the causal agent of the disease, a spore suspension with 106 spores of HlDc1/mL was smeared on healthy leaves of six months old with brushes. Leaves in the control group were smeared with sterile water. All inoculated plants were incubated at 28°C under a 12-h photoperiod in a moisture chamber. The pathogenicity tests were conducted three times with six plants each time. Fifteen days post-inoculation, the leaves inoculated with HlDc1 developed red-brown lesions, whereas the control leaves remained asymptomatic. Isolate HlDc1 was re-isolated from infected leaves. To our knowledge, this is the first report of leaf scorch on H. littoralis in Yunnan province, China, caused by D. curtisii. The results laid the foundation for epidemiological forecasting and scientific control of this disease.

First report of Impatiens necrotic spot orthotospovirus infecting Tulbaghia violacia Harv. in China.

Tulbaghia violacea Harv. indigenous to southern African countries, is an herbaceous perennial bulbous plant belonging to the family Amaryllidaceae. It is a popular garden plant in China. This attractive plant is traditionally used as medicine and repellent (Kubec et al. 2002; Moodley et al. 2015). In June 2021, T. violacea plants showing typical tospovirus-like symptoms of chlorotic rings patterns, were found at the campus of Yunnan University of Chinese Medicine (Fig.S1). Disease incidence was about 11.0% during the field survey. Total RNA was extracted from symptomatic leaves of T. violacea plants using the TRIzol reagent (ambio, Carlsbad, CA). Reverse transcription (RT)-PCR was conducted to identify the virus using RNA extract as the template. The degenerate primers (dTospo-F2 and dTospo-R2) (Huang et al. 2018) were used to amplify the conserved regions of the orthotospoviral L RNA sequences. No amplification was obtained from extracts of two asymptomatic plants. The amplicons from four symptomatic samples were cloned into the pMD19-T vector (TaKaRa) and sequenced (three clones for each amplicon) by Tsingke (Shanghai, China). The obtained DNA fragments were determined to be 312 bp. The sequences from four symptomatic samples were identical (GenBank acc.no. OK258285) and shared the highest nucleotide identities (98.0%) with a corresponding sequence of segment L of impatiens necrotic spot virus (INSV) isolated (GQ336991) from Phalaenopsis amabilis in Yunnan province, China. To further confirm the INSV infection to T. violacea, the samples were analyzed with the specific primers for the N, NSs and NSm genes of INSV (Table S1), respectively. Amplicons of the expected size, 789 bp, 1344 bp and 912 bp, were produced, respectively. Amplicons were cloned and sequenced. The 789-bp N (ON529554) and 1344-bp NSs (ON529554) gene sequences had 99.1% and 99.3% nucleotide identities with the corresponding region of previously described INSV Phalenopsis isolate (GQ336989), respectively. The 912-bp NSm (ON529553) gene sequence shared 99.5% nucleotide identity with the corresponding region of INSV Phalenopsis isolate (GQ336990). Metavirome and Sanger sequencing were used to complete the genome of INSV from T. violacea. The leaves of the symptomatic sample were used to construct an rRNA-depleted library using Nextera XT reagents (Illumina, San Diego, CA). The library was subjected to RNA-Seq a NovaSeq 6000 platform (Illumina, San Diego, CA). A total of 33,193,233 quality-filtered reads were obtained using BBMAP (https://github.com/BioInfoTools/BBMapBBMap - Bushnell B. - sourceforge.net/projects/bbmap/). Among 161052 reads mapped to virus sequences, 151407 reads (read ratios 94.0%) were mapped to INSV. Three complete segments of INSV genome were determined to 8,778 nt (L segment, Acc. No. ON529552), 4,958 nt (M segment, Acc. No. ON529553), and 2,983 nt (S segment, Acc. No. ON529554) in length. These segments were validated by RT-PCR and Sanger sequencing. Three segments share nucleotide sequence identities of 99.6%, 99.3% and 98.9% with the L (GQ336991), M (GQ336990) and S segments (GQ336989) of INSV Phalenopsis isolate, respectively. The results of sequence comparisons showed no evidence of reassortment between INSV and another orthotospovirus. There was a report of tomato spotted wilt virus infecting T. violacea in Florida, USA (Dey et al. 2019). No other virus infecting T. violacea was reported. INSV has been reported to infect several economically important crops including Phalenopsis, pepper etc. in China (Chen et al. 2016). INSV-infected T. violacea not only losses landscaping value but also plays an important intermedia host role in the spread of INSV. Additional surveys and evaluation will be needed to understand the potential medicinal effect of this virus on this plant. To our knowledge, this is first report of INSV in T. violacea.

Preserving hepatic artery flow during portal triad blood occlusion improves regeneration of the remnant liver in rats with obstructive jaundice following partial hepatectomy.

In certain cases, major hepatectomy is essential and inevitable in patients with hilar cholangiocarcinoma and obstructive jaundice (OJ). The current study was designed to evaluate effects of a novel method of portal blood occlusion, where the portal vein was occluded (OPV) and the hepatic artery flow was preserved in rats with OJ that underwent partial hepatectomy. OJ was induced in rats by ligation of the common bile duct for 7 days. Subsequently, OJ rats underwent hepatectomy removing 76% of the liver following occlusion of the portal triad (OPT), OPV or without portal blood occlusion. Liver blood flow (LBF), liver damage and regeneration were assessed. The safety limit for the duration of liver ischemia was 20 min for OPT and 40 min for OPV in rats with OJ. OPT and OPV methods resulted in significantly decreased microvascular LBF in rats with OJ from 529.53±91.55 laser speckle perfusion units (LSPU) in the control to 136.89±32.32 and 183.99±49.25 LSPU, respectively. Liver damage was assessed analyzing levels of serum alanine transaminase and direct bilirubin, determining interleukin-1ß and tumor necrosis factor-α expression and histological examination. It was demonstrated that liver damage and caspase-3 and -9 expression in the liver were substantially reduced in the OPV group compared with the OPT group. In addition, the OPV method significantly improved liver regeneration in OJ rats, as indicated by increased rates of liver regeneration and expression of proliferating cell nuclear antigen and Ki-67 compared with the OPT group. Therefore, the OPV method may prolong the duration of portal blood occlusion, reduce liver injury and improve liver regeneration by preserving hepatic arterial flow during portal blood control in rats with OJ undergoing partial hepatectomy. The current study describes a novel technique, which may be applied in liver surgery in patients with complex jaundice.

Proper bile duct flow, rather than radical excision, is the most critical factor determining treatment outcomes of bile duct cysts.

BACKGROUND: The purpose of this study was to compare the impact of the extent of excision and the patent bile duct flow on treatment outcomes of bile duct cysts (BDCs). METHODS: We retrospectively analyzed the records of 382 patients who received surgery for BDCs from January 2005 to December 2014. RESULTS: For Type Ia cysts, proper bile flow was associated with good long-term treatment outcomes with a greater level of significance (p < 0.001) than complete excision (p = 0.012). For Type IVa cysts, proper bile flow, but not complete excision, was associated with good long-term outcomes (p < 0.00001). In addition, 96.3% (104/108) of Type IVa patients with proper bile flow had no late complications and good biliary function, while no patient without patent bile flow had a good clinical outcome. For Type Ic cysts, 92 patients who received partial excisions had good outcomes when proper bile flow was restored. Regression analysis revealed that the absence of proper bile flow, in comparison to incomplete excision, is a greater risk factor for poor long-term treatment effects for Type Ia and Type IVa cysts. CONCLUSIONS: Compared to complete excision, the establishment of proper bile flow exerted a greater impact on improving long-term clinical outcomes after BDC surgery.

Ex vivo liver resection and autotransplantation as alternative to allotransplantation for end-stage hepatic alveolar echinococcosis.

BACKGROUND & AIMS: Radical resection is the best treatment for patients with advanced hepatic alveolar echinococcosis (AE). Liver transplantation is considered for selected advanced cases; however, a shortage of organ donors and the risk of postoperative recurrence are major challenges. The aim of this study was to assess the clinical outcomes of ex vivo liver resection and autotransplantation for end-stage AE. METHODS: In this prospective study, 69 consecutive patients with end-stage hepatic AE were treated with ex vivo resection and liver autotransplantation between January 2010 and February 2017. The feasibility, safety and long-term clinical outcome of this technique were assessed. RESULTS: Ex vivo extended hepatectomy with autotransplantation was successful in all patients without intraoperative mortality. The median weight of the graft and AE lesion were 850 (370-1,600) g and 1,650 (375-5,000) g, respectively. The median duration of the operation and anhepatic phase were 15.9 (8-24) h and 360 (104-879) min, respectively. Six patients did not need any blood transfusion. Complications higher than IIIa according to Clavien classification were observed in 10 patients. The 30-day-mortality and overall mortality (>90 days) were 7.24% (5/69) and 11.5% (8/69), respectively. The mean hospital stay was 34.5 (12-128) days. Patients were followed-up systematically for a median of 22.5 months (14-89) without recurrence. CONCLUSION: This is the largest series assessing ex vivo liver resection and autotransplantation in end-stage hepatic AE. This technique could be an effective alternative to liver transplantation in patients with end-stage hepatic AE, with the advantage that it does not require an organ nor immunosuppressive agents. LAY SUMMARY: Ex vivo liver resection and autotransplantation were performed in a large series of patients with end-stage hepatic alveolar echinococcosis. The results showed that this surgical option was feasible, with acceptable postoperative mortality, but 100% disease-free survival in survivors. Careful patient selection, as well as precise assessment for size and quality of the remnant liver are key to successful surgery.

In situ splitting after selective partial portal vein ligation or simultaneous hepatic artery ligation promotes liver regeneration.

This study seeks to compare the impact of selective partial portal vein ligation (PPVL) or the combination of simultaneous hepatic artery ligation (PPVAL) with in situ splitting (ISS) on liver regeneration and injury. Rats were randomized into three groups; namely: selective PVL, PPVL + ISS and PPVAL + ISS. The changes in hepatic hemodynamics, liver regeneration and hepatocytic injury were examined. Blood flow to the left portal branch and the microcirculation of the left median lobe after PPVL or PPVAL was significantly reduced. Liver regeneration of PPVAL + ISS group was more pronounced than that in the PPVL + ISS and PVL groups at 48 and 72 hours as well as 7 d postoperatively. The serum biochemical markers and histopathological examination demonstrated reduced levels of liver injury in the PPVL + ISS group. Injury to hepatocytes was more pronounced with PPVAL + ISS than PVL. HGF, TNF-α and IL-6 expression in the regenerated lobes in both PPVAL + ISS and PPVL + ISS groups increased significantly when compared to the PVL group. We demonstrated that both PPVL + ISS and PPVAL + ISS were effective and feasible means of inducing remnant liver hypertrophy and could serve as a rapid clinical application for qualified patients.

Augmented reality technology for preoperative planning and intraoperative navigation during hepatobiliary surgery: A review of current methods.

BACKGROUND: Augmented reality (AR) technology is used to reconstruct three-dimensional (3D) images of hepatic and biliary structures from computed tomography and magnetic resonance imaging data, and to superimpose the virtual images onto a view of the surgical field. In liver surgery, these superimposed virtual images help the surgeon to visualize intrahepatic structures and therefore, to operate precisely and to improve clinical outcomes. DATA SOURCES: The keywords "augmented reality", "liver", "laparoscopic" and "hepatectomy" were used for searching publications in the PubMed database. The primary source of literatures was from peer-reviewed journals up to December 2016. Additional articles were identified by manual search of references found in the key articles. RESULTS: In general, AR technology mainly includes 3D reconstruction, display, registration as well as tracking techniques and has recently been adopted gradually for liver surgeries including laparoscopy and laparotomy with video-based AR assisted laparoscopic resection as the main technical application. By applying AR technology, blood vessels and tumor structures in the liver can be displayed during surgery, which permits precise navigation during complex surgical procedures. Liver transformation and registration errors during surgery were the main factors that limit the application of AR technology. CONCLUSIONS: With recent advances, AR technologies have the potential to improve hepatobiliary surgical procedures. However, additional clinical studies will be required to evaluate AR as a tool for reducing postoperative morbidity and mortality and for the improvement of long-term clinical outcomes. Future research is needed in the fusion of multiple imaging modalities, improving biomechanical liver modeling, and enhancing image data processing and tracking technologies to increase the accuracy of current AR methods.

A study on risk factors and diagnostic efficiency of posthepatectomy liver failure in the nonobstructive jaundice.

Liver failure remains as the most common complication and cause of death after hepatectomy, and continues to be a challenge for doctors.t test and χ test were used for single factor analysis of data-related variables, then results were introduced into the model to undergo the multiple factors logistic regression analysis. Pearson correlation analysis was performed for related postoperative indexes, and a diagnostic evaluation was performed using the receiver operating characteristic (ROC) of postoperative indexes.Differences in age, body mass index (BMI), portal vein hypertension, bile duct cancer, total bilirubin, alkaline phosphatase (ALP), gamma-glutamyl transpeptidase (GGT), operation time, cumulative portal vein occlusion time, intraoperative blood volume, residual liver volume (RLV)/entire live rvolume, ascites volume at postoperative day (POD)3, supplemental albumin amount at POD3, hospitalization time after operation, and the prothrombin activity (PTA) were statistically significant. Furthermore, there were significant differences in total bilirubin and the supplemental albumin amount at POD3. ROC analysis of the average PTA, albumin amounts, ascites volume at POD3, and their combined diagnosis were performed, which had diagnostic value for postoperative liver failure (area under the curve (AUC): 0.895, AUC: 0.798, AUC: 0.775, and AUC: 0.903).Preoperative total bilirubin level and the supplemental albumin amount at POD3 were independent risk factors. PTA can be used as the index of postoperative liver failure, and the combined diagnosis of the indexes can improve the early prediction of postoperative liver failure.

A novel laparoscopic transcystic approach using an ultrathin choledochoscope and holmium laser lithotripsy in the management of cholecystocholedocholithiasis: An appraisal of their safety and efficacy.

BACKGROUND: Although laparoscopic transcystic common bile duct exploration (LTCBDE) is the preferred approach for CBD stone clearance, the success rate can vary between 55% and 85%. This study evaluated if ultrathin choledochoscope and holmium laser lithotripsy could improve the success rate of LTCBDE. METHODS: Records of 126 patients (average age, 46.1 ± 13.8 years) with cholecystocholedocholithiasis treated with laparoscopic cholecystectomy (LC) and LTCBDE were retrospectively reviewed. RESULTS: LC+LTCBDE was performed successfully in 118 of 126 patients, with a surgical success rate of 93.7%. An ultrathin choledochoscope was used in 75 (63.5%) patients, and holmium lithotripsy was performed in 38 (32.2%) patients. The stone clearance rate was 99.2% (117/118). No significant complications occurred. One hundred (84.7%) patients had excellent and 13 (11%) had good outcomes for an overall success rate (excellent plus good) of 95.7%. CONCLUSION: Ultrathin choledochoscope and holmium laser lithotripsy can improve the surgical outcomes of LC+LTCBDE with minimal complications.