Nanobodies are derived from the variable domain of the heavy-chain antibodies (HCAbs) that occur naturally in the serum of Camelidae. They are the smallest antibody fragments capable to bind antigens. With the characteristics of their increased solubility, increased domain stabilities, nanomolar affinities, easy crossing the blood-brain barrier, easy generation, engineering, optimization and tailoring, easy humanization, nanobodies have extensive application prospects in diagnosis and detection. Although nanobody has demonstrated tremendous success, a number of practical challenges limit its broader applications in disease diagnosis and detection, including construction of a phage library and selection of nanobody fragments with high affinity and immunogold labeling technique. Here, we review several recent findings on the use of nanobodies in molecular diagnostics and suggest some practical strategies in resolving the current challenges in this attractive research area, particularly to optimize the affinity, solubility, humanization of nanobodies.
Humans ; Immunoglobulin Heavy Chains ; chemistry ; Single-Domain Antibodies ; chemistry ; drug effects

Objective:To investigate the expression of nicotinamide adenine dinucleotide (NAD +) digestive enzyme CD38 in normal and endotoxin-tolerant human monocyte THP-1 cell lines treated with lipopolysaccharide (LPS). Methods:(1) Normal THP-1 cells: The experiment and control group were treated with 100 ng/ml LPS for 1, 3, 6, 12 and 24 h or phosphate buffer for 24 h, respectively. Quantitative polymerase chain reaction (PCR) and Western blot were used to measure the expression of interleukin-6 (IL-6) and tumor necrosis factor (TNF) mRNA, CD38 mRNA and protein. (2) The induced endotoxin-tolerant THP-1 cells: ①The model of endotoxin-tolerant cells was established firstly by treating the THP-1 cells with 100 ng/ml LPS for 24 h. THP-1 cells treated with phosphate buffer were set as blank group. After further stimulating the two groups with LPS (100 ng/ml) for 3 h, mRNA levels of IL-6 and TNF were measured by quantitative PCR to determine whether the modeling was successful or not. ②In addition, the expression of CD38 mRNA were detected by quantitative PCR before and 12 h after LPS stimulation, and the expression of CD38 protein of these two groups were detected by western blotting before and 1, 6 h after LPS stimulation. Two independent samples t-test and repeated measurement analysis of variance were used for statistical analysis. Results:(1) In normal THP-1 cells, the mRNA expression levels of IL-6 and TNF in the LPS-stimulated cells were significantly higher than those of the control at all time points. And a higher expression level of CD38 mRNA and protein was observed in LPS-stimulated cells from 3 to 24 h compared with the control (mRNA at 3 h: 2.27±0.03 vs 1.00±0.18; protein at 3 h: 1.47±0.14 vs 1.00±0.16, both P<0.05). (2) Endotoxin-tolerant THP-1 cells: ①IL-6 and TNF mRNA levels in the model group were significantly lower than those in the blank group (both P<0.05), indicating that the endotoxin-tolerant THP-1 cell model was established successfully. ②Compared with the same points in the blank group, CD38 mRNA expression was upregulated in the model group before stimulating by LPS (14.18±1.19 vs 1.00±0.13, t=19.007) and 12 h after LPS stimulation (28.33±3.98 vs 7.61±0.88, t=8.803). Moreover, CD38 protein levels before stimulating by LPS (1.54±0.06 vs 1.00±0.10, t=7.796) and 1 h (1.59±0.09 vs 1.07±0.17, t=4.721), 6 h after LPS stimulation (2.48±0.09 vs 1.43±0.12, t=12.233) in the model group were all higher than those in the control group (all P<0.05). The intra-group comparison showed that in the model group the levels of CD38 mRNA at 12 h and CD38 protein at 6 h after LPS stimulation were significantly higher than those before (both P< 0.05). Conclusions:In both normal and endotoxin-tolerant THP-1 cells, LPS upregulates the expression of CD38, which is an NAD + digestive enzyme, and an indirect indicator of NAD + level in monocyte reinfection at the stage of immunosuppression. This study provides a preliminary reference for further investigation on the applicability of CD38 as a potential biological marker in the clinical diagnosis of neonatal sepsis.

Objective:To investigate the etiology of high myopia and the correlation between best corrected visual acuity and myopic diopter and fundus lesions.Methods:The clinical data of 112 patients (198 eyes) with high myopia diagnosed in Yuyao Hospital of Traditional Chinese Medicine from December 2018 to December 2019 were retrospectively analyzed. The factors that influence high myopia were analyzed. Myopic diopter, the type and severity of fundus lesions, and best corrected visual acuity were evaluated. The correlation between observation indexes was analyzed.Results:The occurrence of high myopia was related to partial lens opacity, fundus aberrations, corneal pannus and heredity. Unconditional multivariable logistic regression revealed that parental myopia [73 (83.91%) vs. 39 (57.34%)], outdoor activities < 4.17 h [47 (89.36%) vs. 24 (61.54%), 26 (68.42%), 20 (65.42%)] are the risk factors of high myopia ( χ2 = 13.427, 10.300, P < 0.05 or P < 0.01). Pearson linear correlation analysis showed that myopia diopter was negatively correlated with best corrected visual acuity ( r = -0.729, P < 0.001). Tigroid fundus, tilted optic disc, β-zone parapapillary atrophy and posterior polar annular choroidal dystrophy were positively correlated with best corrected visual acuity ( r = 0.461, 0.732, 0.528, 0.825, P < 0.05 or P < 0.001). The severity of lesion was negatively correlated with best corrected visual acuity( r = -0.673, P < 0.001). Conclusion:The myopia diopter and fundus lesion characteristics in patients with high myopia are closely related to best corrected visual acuity, which can be used to evaluate patient's condition and provide guidance for prevention and treatment of high myopia.

Objective To study the effect of radiofrequency ablation (RFA) with sublethal temperature on the production of liver cancer stem cells (LCSCs) and on the expression of LCSCs-related transcriptional factors.Methods Mouse hepl-6 hepatoma cell line and clinical samples of patients with hepatocellular carcinoma (HCC) were used to test the expressions of LCSCs-related markers and transcriptional factors.Results Different temperatures were used to stimulate Hep1-6 cells,and it was proved that the temperature of 45℃ was a sublethal temperature that could not induce cell death.Flow cytometry testing showed that treatment with 45℃ could obviously increase CD13+,CD44+,CD90 and CD133+ Hep1-6 cells,suggesting that treatment with 45℃ could increase the production of above mentioned types of LCSCs in hep1-6 cells.Real-time quantitative polymerase chain reaction (RT-qPCR) assay indicated that the temperature of 45℃could cause significant increase in CD13,CD90 and CD133 mRNA.In all 5 HCC patients,CD13 mRNA in the recurrent HCC lesions was remarkably increased,CD133 mRNA was increased in 4 patients with recurrent HCC,and CD90 mRNA was increased in only one patient with recurrent HCC.Flow cytometry testing revealed that CD13+ LCSCs were strikingly increased in 4 recurrent HCC patients,while CD133+LCSC was increased in only one patient,suggesting that more close correlation existed between the increase of CD13+ LCSCs and the temperature of 45℃.RT-qPCR assay showed that in 4 recurrent HCC patients with increased CD13+ LCSC,the Sox2 and Stat2 among 13 LCSCs-related transcriptional factors were obviously increased.Flow cytometry testing showed that 45℃ treatment also increased the expression of Sox2 and Stat1 mRNA in Hep1-6 cells.Finally,Sox2 and Stat1 could be knockdown by siRNAs,indicating that both Sox2 and Stat1 transcriptional factors were involved in 45℃-induced production of CD13+ LCSCs in Hep1-6 cells.Conclusion In RFA therapy,the use of sublethal temperature of 45℃ can increase CD13+LCSCs,which is related to the promotion of Sox2 and Stat1 expression.The results of this study can be used for reference in the research of liver cancer recurrence.

[Objective]To evaluate the terminal hair growth of different body positions among in pregnant women ,analyze the contribution of each area to the diagnosis to hirsutism ,to improve the mFG scoring system.[Method]Pregnant women aged 20~41 years,with normal range of total testosterone levels and non-hirsute were recruited at their prenatal care in Sun Yat-sen Memorial Hospital,Sun Yat-sen University. They were followed up before pregnancy,at the 5th and the 9th week,the 10th and the 14th week, the 15th and the 20th week,the 21th and the 24th week of gestation. Then 72 more cases of pregnant women were recruited and followed up at 15~24th week. At each time of followed up,their total testosterone(TT)levels was examined by liquid chromatography tandem mass spectrometry(LC/MS-MS)and terminal hair growth were assessed by mFG score. Significant difference procedure least(LSD) analysis of variance was used to compare the levels of testosterone and mFG score in different gestational weeks. Receiver operating characteristics(ROC)analysis and logistics analysis were conducted to evaluate the contributory strength of hair growth in each body position for the diagnosis of hirsutism. The scores in the body area which made a significant contribution to the total were summed up and termed the simplified mFG score(sFG score). Following,the sFG scores were subjected to ROC analysis to determine the thresh-old that would maximize both the sensitivity and specificity of the measure to accurately distinguish hirsute from non-hirsute patients.[Results]Among the forty three pregnant women who were followed-up from before pregnancy to 24th week,the mean±SD for TT was (1.09 ± 0.59)nmol/L before pregnancy,and(1.13 ± 0.40),(1.28 ± 0.38),(1.83 ± 0.63),(1.82 ± 0.52)nmol/L for 5~ 9th,10~14th,15~20th week,and the mFG score was 1.65 ± 0.60,2.30 ± 0.45,3.60 ± 0.68,4.20 ± 0.41 and 4.40 ± 0.77,respectively. The order of the facial and body sites presented with new terminal hair growth was upper abdomen,lower abdomen,lower back,up-per lip,thighs,upper back,chest,upper arm,and chin,in sequence. After analyzing 115 cases(including the 72 cases recruited later),ROC analysis showed that the diagnostic value of different sites for hirsutism(mFG≥5):upper lip>lower back>thighs>lower abdomen>upper arm>upper back>chest/upper abdomen. Four sites among them ,namely upper lip ,lower back ,thighs and lower abdomen had the best diagnostic value,and the AUC for ROC were 0.779,0.728,0.675,and 0.626,respectively. Both ROC and logistic analysis indicated that he most significant body areas in defining hirsutism(defined as an mFG score≥5)were the upper lip, lower back,thighs,and lower abdomen. Using a cut-off value of 3,the combination of the four areas has the best sensitivity and specificity in distinguishing hirsute from non-hirsute women. [Conclusion]The study suggested that the mFG score increased as pregnancy progressed before the 24 weeks of gestation. The subset of upper lip,lower back,thighs and lower abdomen may be a reli-able simplification of mFG system for the evaluation of excess hair growth. The cut-off value was of≥3.

Objective:To explore indications for replantation of proximal proper digital artery and establishing extrinsic arterial perfusion pressure in the treatment of special type of severed digits with avulsion over 12.0 hours of warm ischemia, and to analyse the factors that affected the survival rate of the replanted digits.Methods:From September 2014 to January 2022, 8 patients with severed digits and prolonged warm ischemia were treated by transposition of adjacent digital artery together with the technique of extrinsic arterial perfusion pressure in the Department of Wrist and Hand Surgery, the Orthopaedic Hospital in Sichuan Province. During the operation, the defected proximal proper digital artery was reconstructed and repaired with vein graft, one side of the digital artery was repaired with an inverted "Y" vein graft, and one side of "Y" vein was bridged and anastomosed to repair the original digital artery. On the other side, the adjacent proximal proper digital arteries were transpositioned and anastomosed to gain an extrinsic arterial perfusion pressure, which increased the distal haemodynamic and reconstituted the blood supply. Of the 8 patients (9 severed digits) : 1 had severed index and middle fingers, 2 had severed index fingers, 4 had severed thumbs and 1 had severed little finger. All the patients were males, aged 16-63 years old, at 37.6 years old in average. Warm ischemia time of the severed digits were 12.3-20.6 hours, with 17.4 hours in average. The survival rate of replanted digits was observed after surgery. Postoperative follow-ups were conducted through telephone or WeChat reviews.Results:Follow-up time was 6-26 months, at 8.3 months in average. Retrospective analysis was performed. Vascular compromises occurred in 3 patients 4 digits (arterial insufficiency in 1 digit, venous congestion in 3 digits), skin necrosis occurred in 1 patient (1 digit) and digit necrosis in 1 patient (1 digit). Overall, 8 of the 9 replanted digits survived. According to the Replantation Function Evaluation Standard of Hand Surgery Association of Chinese Medical Association, the digit function after replantation was evaluated at excellent in 6 digits, good in 1 digit and poor in 1 digit.Conclusion:For a severed digit with an ischemia time over 12.0 hours, the survival rate can be improved by transposition of an adjacent digital artery to provided extrinsic arterial perfusion pressure.

Objective To study the role of partial pressure of oxygen in the treatment of pulmonary hypertension induced by meconium aspiration syndrome. Methods Retrospective analysis was performed on 58 patients with meconium aspirate and pulmonary hypertension from January 1,2007 to December 31,2016, 28 patients in the death group,and 30 patients in the survival group. Blood gas analysis about two groups of children with mechanical ventilation were collected at four different time points:before mechanical ventilation ( 0 h) ,12 hours after mechanical ventilation,24 hours after mechanical ventilation and 72 hours after mechan-ical ventilation. The 80 mmHg(1 mmHg=0. 133 kPa) in blood gas analysis of the partial pressure of oxygen was used as the boundary point to study the effects about different blood oxygen partial pressures on the length of mechanical ventilation time in the survival group. Results Blood gas analysis showed that there were no significant differences in pH,partial pressure of oxygen,HCO3-,and lactic acid before mechanical ventilation in the two groups,but the pH values were significantly higher in the survival group after 12 h, 24 h,and 72 h of mechanical ventilation(t values:-2. 07,-3. 48,-7. 11;P <0. 05). Partial pressure of oxygen were higher in the surviving group at 12 h,24 h,and 72 h than those in the death group(t values-2. 87,-4. 88,-11. 29; P<0. 05). The HCO3-values of the survival group at 12 h,24 h and 72 h were higher than the death group, and the differences were statistically significant ( t values: -3. 90, -5. 60,-5. 76,P<0. 001). The lactic acid of survival group was significantly lower than the death group at 72 h after ventilation (t=5. 12,P<0. 001). Compared with the death group,partial pressure of carbon dioxide in 24 h,72 h decreased significantly in the surviving group(t values:5. 67,7. 60;P<0. 05). In the surviving group,the partial pressure of oxygen was maintained above 80 mmHg in 18 patients and the mechanical venti-lation time was (7. 17 ± 1. 95) d. The partial pressure of oxygen was below 80 mmHg in 12 patients of the surviving group and the mechanical ventilation time was (8. 67 ± 2. 50)d. The difference was statistically sig-nificant(t =12. 02,P <0. 001). Conclusion In the treatment of newborn with pulmonary hypertension caused by meconium aspiration,maintaining a higher pH value and a partial pressure of oxygen higher than 80 mmHg may be beneficial to the recovery of children with pulmonary hypertension.

Objective To explore the clinical effect of reconstructing distal radioulnar ligament with al-lograft tendon under wrist arthroscopy in the treatment of chronic PalmerⅠD type triangular fibrocarti-lage complex injury complicated with distal radioulnar joint instability.Methods Clinical data of 15 pa-tients with chronic PalmerⅠD type triangular fibrocartilage complex injury and admitted to our hospital between January 2017 to January 2020 were retrospectively analyzed.All of them were treated with wrist arthroscopic allograft tendon reconstruction of distal radioulnar ligament.During the postoperative follow-up,the wrist range of motion and grip strength were evaluated,while the wrist pain and wrist function were assessed using the visual analogue scale(VAS)and modified Mayo score.Results All pa-tients were followed up twelve to twenty-four months,with an average of(18.15±2.20)months.The postoperative VAS score was(2.56±0.69),significantly better than the preoperative one(6.78±1.24)(P<0.05).Moreover,the postoperative ratio of grip strength between the affected and the healthy hand was(76.18±9.72)%,significantly better than that of(50.18±6.79)%(P<0.05),with increased grip strength.Meanwhile,the postoperative rotational motion of wrist joint[(151.09±12.38)°]improved significantly compared with that before operation[(142.87±10.19)°](P<0.05).According to the modi-fied Mayo score,8 cases were excellent,6 cases were good,and 1 case was fair,reaching an excel-lent and good rate of 93.33%.Conclusion Wrist arthroscopic reconstruction of distal radioulnar liga-ment with allograft tendon is effective in the treatment of chronic PalmerⅠD type triangular fibrocarti-lage complex injury with lower radioulnar joint instability,relieving pain and promoting grip strength and rotational motion.

Objective: To investigate the genetic characteristics of Lamivudine-resistant mutation patterns and HBV S gene mutants in patients with chronic hepatitis disease of different disease progression. Methods: Blood samples of LAM-resistant patients with chronic hepatitis disease were collected. HBV RT gene nucleotide sequences were obtained, and then differences in drug-resistant mutation patterns, drug susceptibility and HBV S gene mutants characteristics between the two groups were analyzed. Results: Forty-seven chronic hepatitis B (CHB) patients and 16 HBV-related liver cirrhosis (LC)/HBV-related hepatocellular carcinoma (HCC) patients were included in this study. M204I single point mutation and L180M+ M204I/V were the most common pattern during patients with chronic hepatitis disease (35/63, 55.56%). The numbers of resistant to three nucleos(t)ide analogues in LC/HCC group was higher than CHB group’s (62.50% vs 34.04%, P=0.046). In HBV S gene, more immune associated HBsAg-escape mutations were detected in LC/HCC group than that in CHB group (62.50% vs 31.91%, P=0.031). I126T/V and G145A (for LCC/HCC group, 60%), I126S/T and S117T (for CHB group, 46.67%) were showed as the most common form for HBsAg escape mutations in the two groups. The two groups both detected RT mutations concomitantly with stop codon mutations in S gene (rtA181T/sW172* and rtM204I/sW196*). Conclusions Different characteristics in Lamivudine-resistant mutations and associated HBV S gene mutants were found in patients with chronic hepatitis disease of different disease progression, and LC/HCC patients exhibit more multi-drug resistant variants and immune associated HBsAg-escape mutants than CHB patients.

Aggregatibacter aphrophilus is a member of the normal flora of the human oral cavity and pharynx, a Gram-negative fastidous bacteria, belonging to agglomerates, which is a normal mixed oropharyngeal flora in humans, most commonly colonized on the surface of oral mucosa. This bacterial infection is rare in clinical practice, and it is difficult to culture and identify the bacteria, which is easy to be missed. A patient with intracranial infection was admitted to Huaihe Hospital, who showed fever and headache as the main clinical manifestations, and Aggregatibacter aphrophilus was detected by the metagenomic next-generation sequencing of cerebrospinal fluid. The patient′s symptoms were significantly improved after anti-infection treatment.