Role of tear size and tendon degeneration for development of pain in rat models of rotator cuff tear.

BACKGROUND: Rotator cuff tear (RCT) is a frequent etiology of shoulder pain and disability; however, the triggers for the onset and aggravation of pain remain obscure. In this study, we established novel rat RCT models to examine the impact of tear size and tendon degeneration on pain. METHODS: Fifty-five adult male Sprague-Dawley rats were allocated into 4 study groups: large tear (L group, n = 10), small tear (S group, n = 15), small tear with scratching (S+ group n = 15), and sham surgery (Sham group, n = 15). Pain-related behaviors were evaluated by weight distribution of forelimbs during a 5-minute free gait using a dynamic weight-bearing apparatus at 2, 4, 6, and 8 weeks. Calcitonin gene-related peptide (CGRP) expressions in ipsilateral dorsal root ganglion (DRG) neurons of C4, C5, and C6 were evaluated at 4 and 8 weeks. The area of scar tissues around the torn tendon, infiltration of inflammatory cells, and severity of tendon degeneration (modified Bonar score) were histologically assessed at 4 and 8 weeks. Additionally, enzyme-linked immunosorbent assay (ELISA) was conducted to evaluate the levels of cyclooxygenase-2 (COX-2) and nerve growth factor (NGF) expression in torn tendons and surrounding tissues at 4 weeks. RESULTS: The weight distribution ratio (ipsilateral and contralateral side) was significantly decreased in the L and S+ group compared with its baseline and Sham group (P < .05), but the S group showed no significant difference compared with the Sham. The ratio of CGRP-immunoreactive neurons in the DRGs was significantly higher in the L and S+ groups than in the S and Sham groups. The histologic assessment indicated that scar tissue formation was more extensive in the L group than in the S and S+ groups. Still, there was no significant difference between the S and S+ groups. The modified Bonar score was considerably higher in the S+ group than in the S group. Furthermore, ELISA analysis demonstrated no significant disparity in COX-2 levels between the groups; however, NGF levels were substantially higher in the S+ group than in the S and Sham groups. CONCLUSION: The present study provides compelling evidence that large RCT is strongly associated with heightened pain severity in a rat model. Nevertheless, even a small tear can significantly aggravate pain when the torn tendon is degenerated. CGRP upregulation driven by peripheral NGF possibly played a pivotal role in the genesis and exacerbation of pain in small RCT.

Precision of the ultrasonography-integrated tibial extramedullary guide for total knee arthroplasty.

PURPOSE: Precise determination of the tibial mechanical axis in total knee arthroplasty (TKA) requires intraoperative identification of the talus center. We present the ultrasonography-integrated tibial extramedullary guide (USG) that enables real-time visualization of the talus. This study assesses the precision of USG compared to computer-assisted surgery (CAS) and validates the efficacy of USG. METHODS: We evaluated 58 patients (62 knees) who underwent primary TKA retrospectively, categorizing them into USG and CAS groups (31 each). We statistically analyzed demographic data, the preoperative alignment of the tibial plateau to the mechanical axis, the postoperative alignment of the tibial component to planned alignment on 3D-CT, pain visual analog scale, and WOMAC scores. Additionally, the frequency of postoperative outlier from planned alignment over 2° was statistically compared. RESULTS: No significant differences were observed in the preoperative data between the groups. The accuracy (mean deviation from the planned alignment) in both groups was not statistically different. However, in terms of the precision of coronal alignment, the USG group exhibited lower variance than the CAS group in the F-test (F value = 2.76, p = 0.023). Moreover, there were no postoperative deviations beyond 2 degrees in the USG group, in contrast to a 20% outlier frequency in the CAS group (p = 0.024). Concerning the precision of sagittal alignment (variance and deviations over 2°), no statistical differences were identified. CONCLUSION: The USG demonstrated higher precision in the tibial coronal plane than CAS in coronal alignment. Direct identification of the individual talus may enhance precision.

Crystal structures of photosystem II from a cyanobacterium expressing psbA2 in comparison to psbA3 reveal differences in the D1 subunit.

Three psbA genes (psbA1, psbA2, and psbA3) encoding the D1 subunit of photosystem II (PSII) are present in the thermophilic cyanobacterium Thermosynechococcus elongatus and are expressed differently in response to changes in the growth environment. To clarify the functional differences of the D1 protein expressed from these psbA genes, PSII dimers from two strains, each expressing only one psbA gene (psbA2 or psbA3), were crystallized, and we analyzed their structures at resolutions comparable to previously studied PsbA1-PSII. Our results showed that the hydrogen bond between pheophytin/D1 (PheoD1) and D1-130 became stronger in PsbA2- and PsbA3-PSII due to change of Gln to Glu, which partially explains the increase in the redox potential of PheoD1 observed in PsbA3. In PsbA2, one hydrogen bond was lost in PheoD1 due to the change of D1-Y147F, which may explain the decrease in stability of PheoD1 in PsbA2. Two water molecules in the Cl-1 channel were lost in PsbA2 due to the change of D1-P173M, leading to the narrowing of the channel, which may explain the lower efficiency of the S-state transition beyond S2 in PsbA2-PSII. In PsbA3-PSII, a hydrogen bond between D1-Ser270 and a sulfoquinovosyl-diacylglycerol molecule near QB disappeared due to the change of D1-Ser270 in PsbA1 and PsbA2 to D1-Ala270. This may result in an easier exchange of bound QB with free plastoquinone, hence an enhancement of oxygen evolution in PsbA3-PSII due to its high QB exchange efficiency. These results provide a structural basis for further functional examination of the three PsbA variants.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

The organization of the phycobilisome-photosystem I supercomplex depends on the ratio between two different phycobilisome linker proteins.

The phycobilisome (PBS) is an antenna protein complex in cyanobacteria, Glaucocystophytes, and red algae. In the standard PBS, the rod-core PBS, the rods are connected to the core by the rod-core linker protein CpcG. The rod-core PBS transfers the light energy mainly to photosystem (PS) II and to a lesser extent to PSI. Cyanobacteria assemble another type of PBS, the CpcL-PBS, which consists of only one rod. This rod-type PBS is connected to the thylakoid membrane by the linker protein CpcL and is a PSI-specific antenna. In the filamentous heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120, the CpcL-PBS forms a complex with the tetrameric PSI (PBS-PSI supercomplex). The CpcL-PBS and the rod part of the rod-core PBS are identical except for the linker proteins CpcL and CpcG. How cells control the accumulation of the two different types of PBS is unknown. Here, we analyzed two mutant strains which either lack the major rod-core linker CpcG4 or overexpress the rod-membrane linker CpcL. In both mutant strains, more and larger PBS-PSI supercomplexes accumulated compared to the wild type. Our results suggest that CpcL and CpcG4 compete for the same phycobiliprotein pool, and therefore the CpcL/CpcG4 ratio determines the levels of PBS-PSI supercomplexes. We propose that the CpcL-PBS and the rod-core PBS fulfill distinct functions in light harvesting.

Additional benefit of ultrasonography to evaluate nerve root condition of degenerative cervical spine disease.

STUDY DESIGN: A cross-sectional explanatory study. OBJECTIVES: To clarify the nerve root condition and the association between nerve root cross-sectional area (CA) on ultrasonography (US) and other examinations such as MRI or compound muscle action potentials (CMAPs) in degenerative cervical spine diseases. SETTING: A university hospital in Japan. METHODS: Fifty-one patients diagnosed with proximal cervical spondylotic amyotrophy (CSA) (13 patients), cervical radiculopathy of C5 or C6 nerve root (CR) (26 patients), or cervical spondylotic myelopathy (CSM) (12 patients), and twenty-nine healthy volunteers were included in this study. Neurological findings, US findings and CMAPs of deltoid and biceps muscles of all participants were evaluated. In addition, CSA, CR, and CSM patients underwent MRI. RESULTS: A significant correlation was not observed between CA and CMAP amplitude or foraminal diameter on MRI (P > 0.05). In the US examination, the C6 CA of the affected side of the CR group was significantly larger than that of both the normal side and the other groups (P < 0.001). The C5 CA of the affected side of the CSA group clearly showed a bimodal distribution: enlarged and small CA groups. In the CMAP findings, CSA cases respectively showed the lower amplitude of deltoid and bicep CMAPs on both the normal and the affected side (P ≦ 0.01). CSM and healthy volunteers were nearly identical in CA and CMAPs. CONCLUSION: Utilizing US in addition to NCS and MRI can contribute towards an evaluation of the nerve root condition of degenerative cervical spine disease. SPONSORSHIP: no sponsorship.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Arthroscopic labral repair combined with less invasive open-shelf acetabuloplasty for patients with developmental dysplasia of the hip.

Hip arthroscopy has become widely used for intra-articular lesions, such as labral tears and femoral acetabular impingement. However, its use in patients with developmental dysplasia of the hip (DDH) has been controversial and has historically demonstrated mixed results, as acetabular dysplasia may cause instability due to insufficient bony coverage of the femoral head, thus causing excessive stress on the repaired labrum and cartilage. We devised a combined hip arthroscopic labral repair and a less invasive open-shelf procedure using a small skin incision as an anterolateral portal in hip arthroscopy. This novel procedure may improve the stability of the repaired labrum with a bony covering in a minimally invasive manner. Moreover, the shelf procedure can be performed under direct vision in a comparatively safe and precise manner. In total, 13 hips with DDH underwent the procedure for labral tears. All patients were females, with a mean age of 30 years. The mean follow-up period was 33 months. The mean Harris hip score improved from 74.2 to 93.6, and Oxford Hip score improved from 32.4 to 19.3. According to the Tönnis classification, the grade of arthritis preoperatively was grade 0 for nine hips and grade I for four hips. No radiographic progression of osteoarthritis was observed. It is possible that this novel procedure could be an effective treatment for labral tears with DDH and may prevent the early onset of secondary osteoarthritis. In this technical tip, we describe hip arthroscopic labral repair combined with a less invasive open-shelf acetabuloplasty in further detail.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

Prevalence of iliopsoas bursitis in patients with end-stage hip osteoarthritis.

OBJECTIVES: Iliopsoas bursitis (IB) is a relatively rare condition that is associated with hip diseases. It can cause neurological symptoms and swelling of the lower extremities by compressing the femoral nerve and vessels. The aim of this study is to examine the prevalence of IB in patients with end-stage hip osteoarthritis. METHODS: A total of 544 patients underwent total hip arthroplasty between May 2010 and May 2019. All patients were examined using computed tomography (CT) to perform preoperative planning. We reviewed the CT images and examined the prevalence and size of IB. These lesions were divided into three types based on their shape (round type, oval type, and heart-shaped type). RESULTS: Of the 544 patients, IB was found in 37 patients. We observed the round type in 4 patients, the oval type in 31 patients, and the heart-shaped type in 2 patients. Two patients showed severe swelling in the legs and had a blood circulatory disorder of the legs. Both cases were heart-shaped bursitis. CONCLUSION: Symptomatic IB was observed in two cases, both of which had a heart shape surrounding the iliopsoas tendon and femoral neurovascular bundle. Although symptomatic IB is a rare condition, special attention is required for heart-shaped IB.

Efficacy and safety of magnetic resonance-guided focused ultrasound treatment for refractory chronic pain of medial knee osteoarthritis.

OBJECTIVE: To elucidate the efficacy and safety of MRgFUS in the treatment for refractory pain derived from medial knee OA. METHODS: Twenty patients with medial knee OA eligible for total knee arthroplasty were included in this prospective, non-controlled study (UMIN000010193). MRgFUS treatment was provided at the site of most severe tenderness around the medial femorotibial joint of each patient under real-time monitoring of temperature. The goal temperature of the targeted bone surface was 55 °C. Numerical rating scale (NRS) worst pain scores, Western Ontario and McMaster Universities osteoarthritis index (WOMAC) scores, EuroQol 5 dimensions index (EQ-5D) scores and pressure pain threshold (PPT) were evaluated before treatment (baseline) and at 1 week and 1, 3, 6, and 12 months post-treatment, respectively. Complications and adverse events were also assessed clinically and radiographically. RESULTS: Treatment response (a 50% or greater decrease in NRS score) was seen in 14 patients (14/19, 73.7%) at 12 months post-treatment. Mean NRS score rapidly decreased at 1 month after treatment and continued to decline through the following 12 months. At final follow-up, mean NRS score was 3.2 ± 1.9, significantly lower than at baseline (p = 0.0013). Mean WOMAC and EQ-5D scores also improved significantly from 1 month after treatment. Fifteen patients showed significant sustained increases in PPTs at the sites of most severe tenderness. No serious adverse events were observed during and after treatment. CONCLUSIONS: MRgFUS treatments were effective not only for managing refractory pain, but also for improving physical functions without adverse events in elderly patients with medial knee OA.

Chronic postsurgical pain after total knee arthroplasty: A prospective cohort study in Japanese population.

OBJECTIVES: To elucidate the prevalence and risk factors of chronic postsurgical pain (CPSP) after primary total knee arthroplasty (TKA) in Japanese population. METHODS: Consecutive patients undergoing primary TKA in a Japanese tertiary hospital (211 knees) were assessed. CPSP after TKA was defined as moderate to severe pain (VAS >30 mm), either at rest or during walking, one year after surgery. Clinical and radiographic data were compared between CPSP and non-CPSP groups and multivariate logistic regression was used to identify predictors of CPSP. RESULTS: The prevalence of CPSP was 8.8%. CPSP group showed significantly higher preoperative WOMAC subscales (pain, function and stiffness), higher rate of postoperative coronal malalignment (femorotibial angle >178° or <170°) and larger varus angle of tibial component compared with non-CPSP group. Logistic regression analysis revealed that preoperative higher WOMAC pain and postoperative coronal malalignment were independent risk factors of CPSP. In a subgroup analysis of patients with well-aligned TKA, preoperative pain VAS at rest was the only risk factor of CPSP. CONCLUSION: Preoperative severe pain and postoperative coronal malalignment were independent risk factors of CPSP after TKA. Preoperative pain management in patients with severe pain and good coronal alignment after TKA possibly minimize the development of CPSP.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system.

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES-2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose-mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD-like tlr0903 (xcsB) and endoglucanase-like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose-mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean.

The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths but show little or no response to the more attenuated red/far-red wavelengths. Here, we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared with other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum, and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs.

Discrepancy between functional recovery and cutaneous silent period change in surgically treated degenerative cervical myelopathy: a prospective pilot study.

STUDY DESIGN: Exploratory research OBJECTIVES: Cutaneous silent periods (CSPs) that reflect the inhibitory spinal cord reflex, can sensitively detect spinal cord dysfunction, and contribute to the diagnosis of degenerative cervical myelopathy (DCM). However, CSP changes after DCM surgery related to functional improvement have not been reported. SETTING: University hospital in Nankoku, Japan METHODS: CSP recorded at four time points-before surgery, 3, 6, 12 months after surgery-were investigated in 31 hands of 16 DCM patients. CSPs were categorized as follows: normal, delayed onset latency, shortened duration, onset delay with shortened duration, and absent CSP. Myelopathic symptoms were evaluated by the Japanese Orthopaedic Association score (JOA score). RESULTS: Normal CSPs were observed in five hands (16%) before surgery and six hands (19%) twelve months after surgery (P > 0.05). Either onset delay or shortened duration or both were observed in 18 hands (58%) before surgery and 16 hands (52%) twelve months after surgery (P > 0.05). Absent CSPs were observed in eight hands (26%) before surgery and nine hands (29%) twelve months after surgery (P > 0.05). Measured values of onset latency and duration also did not change throughout the study period (P > 0.05). On the other hand, JOA scores improved after surgery. (P = 0.003). CONCLUSIONS: CSP abnormalities persisted after surgery in most cases, indicating irreversible damage of the intramedullary reflex circuit. JOA score recovery without CSP recovery provides insight into postoperative neural recovery in DCM.

Additional benefit of local infiltration of analgesia to femoral nerve block in total knee arthroplasty: double-blind randomized control study.

PURPOSE: Multimodal analgesia has become an important concept in current pain management following total knee arthroplasty (TKA). However, controversy remains over what is the most accepted combination. In this study, the additional benefits of local infiltration of analgesia to femoral nerve block were evaluated. METHODS: Forty patients were randomly allocated into a combined local infiltration of analgesia and femoral nerve block or femoral nerve block alone group. In the former, analgesic drugs consisting of ropivacaine and dexamethasone were injected into the peri-articular tissues, while the same amount of saline was injected into the femoral nerve block group. The primary outcome measure was the total amount of fentanyl consumption by the patient-controlled analgesia pump during the 48-h post-operative period. RESULTS: A combination of local infiltration of analgesia and femoral nerve block had less total fentanyl consumption and a larger knee ROM at post-operative day 2 than femoral nerve block alone (p < 0.05). C-reactive protein levels in the combined treatment group were significantly lower than the femoral nerve block group at post-operative day 3 (p < 0.01). There was no difference between the two groups, post-operatively, on the visual analogue scale for pain at rest or while walking, quadriceps strength, timed up and go test, circumference of thigh, Knee Society Score, and Western Ontario and McMaster Universities Osteoarthritis Index. CONCLUSION: The addition of local infiltration of analgesia to femoral nerve block promoted post-operative pain relief and the recovery of knee ROM in the early post-operative period. This combination is an effective method for post-operative pain management after TKA. LEVEL OF EVIDENCE: Randomized controlled trial, Level I.

Protein Engineering of Dual-Cys Cyanobacteriochrome AM1_1186g2 for Biliverdin Incorporation and Far-Red/Blue Reversible Photoconversion.

Cyanobacteria have cyanobacteriochromes (CBCRs), which are photoreceptors that bind to a linear tetrapyrrole chromophore and sense UV-to-visible light. A recent study revealed that the dual-Cys CBCR AM1_1186g2 covalently attaches to phycocyanobilin and exhibits unique photoconversion between a Pr form (red-absorbing dark state, λmax = 641 nm) and Pb form (blue-absorbing photoproduct, λmax = 416 nm). This wavelength separation is larger than those of the other CBCRs, which is advantageous for optical tools. Nowadays, bioimaging and optogenetics technologies are powerful tools for biological research. In particular, the utilization of far-red and near-infrared light sources is required for noninvasive applications to mammals because of their high potential to penetrate into deep tissues. Biliverdin (BV) is an intrinsic chromophore and absorbs the longest wavelength among natural linear tetrapyrrole chromophores. Although the BV-binding photoreceptors are promising platforms for developing optical tools, AM1_1186g2 cannot efficiently attach BV. Herein, by rationally introducing several replacements, we developed a BV-binding AM1_1186g2 variant, KCAP_QV, that exhibited reversible photoconversion between a Pfr form (far-red-absorbing dark state, λmax = 691 nm) and Pb form (λmax = 398 nm). This wavelength separation reached 293 nm, which is the largest among the known phytochrome and CBCR photoreceptors. In conclusion, the KCAP_QV molecule developed in this study can offer an alternative platform for the development of unique optical tools.

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase.

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.

The Non-Mendelian Green Cotyledon Gene in Soybean Encodes a Small Subunit of Photosystem II.

Chlorophyll degradation plays important roles in leaf senescence including regulation of degradation of chlorophyll-binding proteins. Although most genes encoding enzymes of the chlorophyll degradation pathway have been identified, the regulation of their activity has not been fully understood. Green cotyledon mutants in legume are stay-green mutants, in which chlorophyll degradation is impaired during leaf senescence and seed maturation. Among them, the soybean (Glycine max) green cotyledon gene cytG is unique because it is maternally inherited. To isolate cytG, we extensively sequenced the soybean chloroplast genome, and detected a 5-bp insertion causing a frame-shift in psbM, which encodes one of the small subunits of photosystem II. Mutant tobacco plants (Nicotiana tabacum) with a disrupted psbM generated using a chloroplast transformation technique had green senescent leaves, confirming that cytG encodes PsbM. The phenotype of cytG was very similar to that of mutant of chlorophyll b reductase catalyzing the first step of chlorophyll b degradation. In fact, chlorophyll b-degrading activity in dark-grown cytG and psbM-knockout seedlings was significantly lower than that of wild-type plants. Our results suggest that PsbM is a unique protein linking photosynthesis in presenescent leaves with chlorophyll degradation during leaf senescence and seed maturation. Additionally, we discuss the origin of cytG, which may have been selected during domestication of soybean.