Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell.

Protein stability is often a limiting factor in the development of commercial proteins and biopharmaceuticals, as well as for biochemical and structural studies. Unfortunately, identifying stabilizing mutations is not trivial since most are neutral or deleterious. Here we describe a high-throughput colony-based stability screen, which is a direct and biophysical read-out of intrinsic protein stability in contrast to traditional indirect activity-based methods. By combining the method with a random mutagenesis procedure, we successfully identify thermostable variants from 10 diverse and challenging proteins, including several biotechnologically important proteins such as a single-chain antibody, a commercial enzyme and an FDA-approved protein drug. We also show that thermostabilization of a protein drug using our approach translates into dramatic improvements in long-term stability. As the method is generic and activity independent, it can easily be applied to a wide range of proteins.

[Study on needling depth and direction from different acupoints to sphenopalatine ganglion].

OBJECTIVE: To observe and survey the location of Xiaguan (ST 7), "Die'e" and Quanliao (SI 18) on the surface, and the needling depth and direction from the 3 points to sphenopalatine ganglion. METHODS: Fifteen corpses (30 sides) of adult male were fixed by 10% formalin. The lateral areas of face were dissected from the surface to the deep on the 3 acupoints: the electric drill with the kirschner wire punctured towards the sphenopalatine ganglion and extended to the contralateral areas according to different directions of puncturing sphenopalatine ganglion from the 3 acupoints. The corresponding puncturing points of the 3 acupoints were measured by the coordinate location method. RESULTS: (1) Surface location: the distance between Quanliao (SI 18) and "Die'e" was 21 mm and the distance between Xiaguan (ST 7) and "Die'e" was 17 mm; (2) Inserting depth of each point to sphenopalatine ganglion: the depths of Xiaguan (ST 7), "Die'e" and Quanliao (SI 18) were 49.9 mm, 46.9 mm and 46.6 mm, respectively; (3) The coordinate location of the corresponding puncturing points: the puncturing direction of Xiaguan (ST 7) was anterointernal upper corresponding to the area of connecting center between contralateral Taiyang (EX-HN 5) and Tongziliao (GB 1), the distance between the corresponding inserting point of Xiaguan (ST 7) and Sizhukong (TE 23) was 17.6 mm; the puncturing direction of "Die'e" point was posterointernal upper, and the horizontal distance from the corresponding puncture point to the zygomatic arch was 33 mm and the vertical distance from the corresponding puncture point to the eyes' outer canthus was 42 mm; the puncturing direction of Quanliao (SI 18) was posteriointernal upper and the distance between the corresponding inserting point and the area of contralateral parietal tuber, the distance between the corresponding inserting point of Quanliao (SI 18) and the connecting line of bilateral external acoustic pore was 28 mm, the distance between the corresponding inserting point of Quan-liao (SI 18) and the medial line of the head was 62 mm. CONCLUSION: Understanding the surface location, inserting depths and the general puncturing directions of the 3 points can provide basis for puncturing the sphenopalatine ganglion in clinical practice.

[Clinical anatomy study on the acupuncture safety of tiantu (CV 22) and qishe (ST 11)].

OBJECTIVE: To study on the relation between the regional anatomy and safety of acupuncture at Tiantu (CV 22) and Qishe (ST 11). METHODS: In forty-six adult corpses, 92 sides were dissected to observe the partial anatomy structure of acupuncture path of Tiantu (CV 22) and Qishe (ST 11) and structure characteristics by the steel needle marked method and lay-by-lay dissection method. RESULTS: The bilateral the pleura returning lines behind the manubrium sterni interacted at the sternal angle plane accounted for 50.0% of the total specimens and at the first ribs plane accounted for 6.5% of the specimens; for needling the point Tiantu (CV 22), left brachiocephalic vein was at the same level or close to the manubrium sterni upper fringe in 43.5% of the specimens, the left brachiocephalic vein and the middle of manubrium sterni were at the same level in 56.5% of the specimens; for needling the point Qishe (ST 11), in 68 sides of the specimens, internal jugular vein were pierced, accounting for 73.9%, and in 24 sides of the specimens the left common carotid artery were pierced, accounting for 26.1%; in 50 sides of specimens the vagus nerve were touched by the steel needle, accounting for 54.3. CONCLUSION: In acupuncture of Tiantu (CV 22) and Qishe (ST 11), the needle not only easily injure the upper pleural cavity, but also damage the big blood vessel and the vagus nerve in the mediastinum and the cervical root.

[Effect of skull acupuncture and scalp acupuncture on serum vascular endothelial growth factor in the patient of acute cerebral infarction].

OBJECTIVE: To investigate effect of skull suture acupuncture (skull acupuncture) and scalp acupuncture on serum vascular endothelial growth factor (VEGF) in the patient of acute cerebral infarction (CI). METHODS: Twenty cases of CI were treated with skull suture acupuncture at coronal suture, sagittal suture, lambdoid suture, etc. combined with medication (group B), group C (n=20) with scalp acupuncture at contralateral Dingnie Qian-xiexian (MS 6) and Dingnie Houxiexian (MS 7) plus medication, and group A (n=20) with medication. Changes of serum VEGF contents were investigated in the three groups. RESULTS: After treatment, the serum VEGF content did not significantly change in group A (P > 0.05), and significantly increased in group B and group C (P < 0.01, P < 0.05), with significant difference as group B and C compared with that of group A (P < 0.05), and with no significant difference between group B and group C (P > 0.05). CONCLUSION: Skull suture acupuncture combined with medication and scalp acupuncture plus medication have a similar effect on serum VEGF in the patient of acute cerebral infarction.

[Clinically anatomic study on the relation between the form of pleural cupula and its surrounding commonly-used acupoints].

OBJECTIVE: To explore the relation of the form of pleural cupula of the normal adult with safety of acupuncture at commonly-used acupoints around the pleural cupula. METHODS: The safe depth for the commonly-used acupoints around the pleural cupula and the relation with the form of pleural cupula were investigated in 46 adult corpses with small Kirschner wire location and arrangement dissection. RESULTS: The width of the pleural cupula projection equal to clavicle medial 1/3 accounted for 32. 6% of all the corpses, and the width of the pleural cupula projection more than clavicle medial 1/3 accounted for 59. 8% of all the corpses, the width of the pleural cupula projection less than clavicle medial 1/3 and pleural cupula medial margin located at the sternoclavicular joint medial accounted for 7.6% of all the corpses. The observed points such as Tiantu (CV 22), Qishe (ST 11), Jianjing (GB 21), Dingchuan (EX-B1), Dazhu (BL 11) which were considered be not related to the pleural cupula. When acupuncture is carried out according to criteria of acupoint location and needling direction, and the needle exceeded a limit, the pleural menbrane will be broken and induce destruction. CONCLUSION: Position and form of the pleural cupula have anatomical relation to acupuncture accident for needling the points around the superior pleural cupula, which should be played attention to.

Effects of acupuncture on myelogenic osteoclastogenesis and IL-6 mRNA expression.

The effect of acupuncture and moxibustion on myelogenic osteoclastogenesis and IL-6 mRNA expression is investigated. The result turns to be that the number of myelogenic osteoclasts in the model group is obviously bigger than in the sham operation group (P<0.01), and that in the acupuncture group is markedly smaller than in the model group (P<0.01). The IL-6 mRNA expression in the marrow cells of the model group is significantly elevated, as compared with that in the sham operation group (P<0.01), while the its elevation is lower in the acupuncture group than in the model group (P<0.01). It is therefore concluded that 1) acupuncture effectively modulates the secretion of and then to reduces IL-6 mRNA expression and 2) acupuncture decreases the number of myelogenic osteoclasts.