Medial entorhinal cortex mediates learning of context-dependent interval timing behavior.

Episodic memory requires encoding the temporal structure of experience and relies on brain circuits in the medial temporal lobe, including the medial entorhinal cortex (MEC). Recent studies have identified MEC 'time cells', which fire at specific moments during interval timing tasks, collectively tiling the entire timing period. It has been hypothesized that MEC time cells could provide temporal information necessary for episodic memories, yet it remains unknown whether they display learning dynamics required for encoding different temporal contexts. To explore this, we developed a new behavioral paradigm requiring mice to distinguish temporal contexts. Combined with methods for cellular resolution calcium imaging, we found that MEC time cells display context-dependent neural activity that emerges with task learning. Through chemogenetic inactivation we found that MEC activity is necessary for learning of context-dependent interval timing behavior. Finally, we found evidence of a common circuit mechanism that could drive sequential activity of both time cells and spatially selective neurons in MEC. Our work suggests that the clock-like firing of MEC time cells can be modulated by learning, allowing the tracking of various temporal structures that emerge through experience.

Functional studies on tandem carbohydrate-binding modules of a multimodular enzyme possessing two catalytic domains.

Although functional studies on carbohydrate-binding module (CBM) have been carried out extensively, the role of tandem CBMs in the enzyme containing multiple catalytic domains (CDs) is unclear. Here, we identified a multidomain enzyme (Lc25986) with a novel modular structure from lignocellulolytic bacterial consortium. It consists of a mannanase domain, two CBM65 domains (LcCBM65-1/LcCBM65-2), and an esterase domain. To investigate CBM function and domain interactions, full-length Lc25986 and its variants were constructed and used for enzymatic activity, binding, and bioinformatic analyses. The results showed that LcCBM65-1 and LcCBM65-2 both bind mannan and xyloglucan but not cellulose or ß-1,3-1,4-glucan, which differs from the ligand specificity of reported CBM65s. Compared to LcCBM65-2, LcCBM65-1 showed a stronger ligand affinity and a preference for acetylation sites. Both CBM65s stimulated the enzymatic activities of their respective neighboring CDs against acetylated mannan, but did not contribute to the activities of the distal CDs. The time course of mannan hydrolysis indicated that the full-length Lc25986 was more effective in the complete degradation of mixed acetyl/non-acetyl substrates than the mixture of single-CD mutants. When acting on complex substrates, LcCBM65-1 not only improved the enzymatic activity of the mannanase domain, but also directed the esterase domain to the acetylated polysaccharides. LcCBM65-2 adopted a low affinity to reduce interference with the catalysis of the mannanase domain. These results demonstrate the importance of CBMs for the synergism between the two CDs of a multidomain enzyme and suggest that they contribute to the adequate degradation of complex substrates such as plant cell walls. IMPORTANCE: Lignocellulolytic enzymes, particularly those of bacterial origin, often harbor multiple carbohydrate-binding modules (CBMs). However, the function of CBM multivalency remains poorly understood. This is especially true for enzymes that contain more than one catalytic domain (CD), as the interactions between CDs, CBMs, and CDs and CBMs can be complex. Our research demonstrates that homogeneous CBMs can have distinct functions in a multimodular enzyme. The tandem CBMs coordinate the CDs in catalytic conflict through their differences in binding affinity, ligand preference, and arrangement within the full-length enzyme. Additionally, although the synergism between mannanase and esterase is widely acknowledged, our study highlights the benefits of integrating the two enzymes into a single entity for the degradation of complex substrates. In summary, these findings enhance our understanding of the intra-synergism of a multimodular enzyme and emphasize the significance of multiple CBMs in this context.

Lipase in-situ immobilized in covalent organic framework: Enzymatic properties and application in the preparation of 1, 3-dioleoyl-2-palmitoylglycerol.

In this study, the critical importance of designing an appropriate immobilized carrier and method for free lipase to ensure exceptional biological catalytic activity and stability was emphasized. Covalent organic frameworks (COF-1) were synthesized as a novel porous carrier with an azine structure (-CN-NC-) through the condensation of hydrazine hydrate and benzene-1,3,5-tricarbaldehyde at room temperature. Simultaneously, Rhizomucor miehei lipase (RML) was immobilized within the COF-1 carrier using an in-situ aqueous phase method. Characterization of the carrier and RML@COF-1 and evaluation of the lipase properties of RML and RML@COF-1 through p-Nitrophenyl palmitate hydrolysis were conducted. Additionally, application in the synthesis of 1, 3-dioleoyl-2-palmitoylglycerol (OPO) was explored. The results showed that RML@COF-1 exhibited a high enzymatic loading of 285.4 mg/g. Under 60℃ conditions, the activity of RML@COF-1 was 2.31 times higher than that of free RML, and RML@COF-1 retained 77.25% of its original activity after 10 cycles of repeated use, indicating its excellent thermal stability and repeatability. Under the optimal conditions (10%, 1:8 PPP/OA, 45℃, 5 h), the yield of OPO reached 47.35%, showcasing the promising application prospects of the novel immobilized enzyme synthesized via in-situ aqueous phase synthesis for OPO preparation.

Medial entorhinal cortex plays a specialized role in learning of flexible, context-dependent interval timing behavior.

Episodic memory requires encoding the temporal structure of experience and relies on brain circuits in the medial temporal lobe, including the medial entorhinal cortex (MEC). Recent studies have identified MEC 'time cells', which fire at specific moments during interval timing tasks, collectively tiling the entire timing period. It has been hypothesized that MEC time cells could provide temporal information necessary for episodic memories, yet it remains unknown whether MEC time cells display learning dynamics required for encoding different temporal contexts. To explore this, we developed a novel behavioral paradigm that requires distinguishing temporal contexts. Combined with methods for cellular resolution calcium imaging, we find that MEC time cells display context-dependent neural activity that emerges with task learning. Through chemogenetic inactivation we find that MEC activity is necessary for learning of context-dependent interval timing behavior. Finally, we find evidence of a common circuit mechanism that could drive sequential activity of both time cells and spatially selective neurons in MEC. Our work suggests that the clock-like firing of MEC time cells can be modulated by learning, allowing the tracking of various temporal structures that emerge through experience.